Spatial analysis of DNA methylation

ABSTRACT

Provided herein are methods of identifying a methylation status of an analyte in a biological sample. Also provided herein are methods that combine identifying the methylation status with spatial technology to identify the location of a methylation status in a biological sample.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. patent applicationSer. No. 17/573,119, filed Jan. 11, 2022, which is a continuation ofInternational Application PCT/US2021/039103, with an internationalfiling date of Jun. 25, 2021, which claims priority to U.S. ProvisionalPatent Application No. 63/044,042, filed Jun. 25, 2020, and U.S.Provisional Patent Application No. 63/128,783, filed Dec. 21, 2020. Theentire contents of the foregoing applications are incorporated herein byreference.

BACKGROUND

Cells within a tissue of a subject have differences in cell morphologyand/or function due to varied analyte levels (e.g., gene and/or proteinexpression) within the different cells. The specific position of a cellwithin a tissue (e.g., the cell's position relative to neighboring cellsor the cell's position relative to the tissue microenvironment) canaffect, e.g., the cell's morphology, differentiation, fate, viability,proliferation, behavior, and signaling and cross-talk with other cellsin the tissue.

Spatial heterogeneity has been previously studied using techniques thatonly provide data for a small handful of analytes in the context of anintact tissue or a portion of a tissue, or provide a lot of analyte datafor single cells, but fail to provide information regarding the positionof the single cell in a parent biological sample (e.g., tissue sample).

DNA methylation is a crucial epigenetic modification of the genome thatis involved in regulating many cellular processes. These processesinclude, but are not limited to, embryonic development, transcription,chromosome activation, chromosomal stability and as such DNAmethylation, and also aberrant DNA methylation, has been associated withhuman diseases.

DNA methylation is an epigenetic mark that can be inherited throughmultiple cell divisions. However, changes in methylation status can alsooccur in a single cell (e.g., a cancer cell). Thus, in a pathologicalsetting, the methylation state of each DNA molecule can vary, making itsanalysis challenging and cumbersome. This is further confounded by thefact that methylated-cytosine bases are not distinguishable fromunmethylated-cytosine bases in standard DNA sequencing technologies. Tostudy DNA methylation, many researchers rely on the use of bisulfite: achemical which converts unmethylated cytosines to thymines and leavesmethylated cytosines intact. Ideally, bisulfite conversion could be usedfor single-cell technologies, however, the chemical is harsh, fragmentsDNA, and is often not compatible with enzymes and reagents required indownstream steps. However, due to the diagnostic and therapeuticimplications related to targeting DNA methylation, there remains a needto develop reliable and cost-effective methods to ascertain themethylation status of nucleic acids at spatial locations of a biologicalsample.

SUMMARY

This disclosure provides methods of spatial analysis with identificationof DNA methylation status. The methods provided herein are applicable tonormal physiological conditions, development and stem-cell studies, andin pathophysiological settings such as cancer. For example, in oneembodiment, the DNA methylation status (and changes of the same) of oneor more particular analytes can be examined during therapeutic delivery.And, because the location of the analyte (or complement thereof) can beidentified using spatial analysis methods (e.g., RNA-templated ligation)as disclosed herein, the location of the analyte and/or changes to themethylation status can be identified. In some instances, the methodsdisclosed herein can also be combined with imaging techniques thatprovide a correlation between a particular location of an image (e.g.,location of a tumor in a biological sample) and both gene expression andmethylation status at that location. Thus, the disclosure providespowerful methods of identifying the location of methylated DNA in abiological sample.

Accordingly, described herein is a method for identifying methylationstatus of an analyte in a biological sample, the method comprising: (a)contacting the biological sample with an array comprising a plurality ofcapture probes, wherein a capture probe in the plurality of captureprobes comprises (i) a spatial barcode and (ii) a capture domaincomprising at least one methylated cytosine; (b) deaminating the analytein the biological sample; (c) contacting the analyte with a plurality ofprobes comprising a first probe and a second probe, wherein the firstprobe comprises (i) a sequence complementary to at least a firstsequence of the analyte and (ii) a sequence complementary to the capturedomain; and the second probe comprises a sequence complementary to atleast a second sequence of the analyte; (d) ligating the first probe andthe second probe, thereby generating a ligation product; (e) hybridizingthe ligation product to the capture probe; (f) extending the captureprobe using the ligation product as a template; thereby generating anextended capture probe; and (g) determining (i) all or a portion of thesequence of the spatial barcode or the complement thereof, and (ii) allor a portion of the sequence of the extended capture probe, or acomplement thereof, and using the determined sequences of (i) and (ii)to identify the methylation status of the analyte in the biologicalsample.

In some embodiments, the first oligonucleotide and the secondoligonucleotide hybridize to the analyte at sequences that are at leastabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides apart.

In some embodiments, the method described herein further comprisesextending the first oligonucleotide and/or the second oligonucleotideafter step (c). In some embodiments, the first probe and/or second probeis extended using a polymerase.

In some embodiments, the method described herein further comprisesreleasing the ligation product from the analyte. In some embodiments,the releasing occurs before hybridizing the ligation product to thecapture probe.

In some embodiments, the method further comprises, prior to step (b),contacting the biological sample with a permeabilization reagent.

In some embodiments, the first probe and/or the second probe comprises asequence that binds to a sequence on the analyte that does not have a CGdinucleotide.

In some embodiments, the method further comprises washing the biologicalsample between step (b) and step (c).

In another aspect, described herein is a method for identifying amethylation status of DNA in a biological sample, the method comprising:(a) providing one or more transposomes to the biological sample, whereinthe transposomes comprise methylated adaptors, under conditions whereinthe one or more methylated adaptors is inserted into the DNA, therebygenerating tagmented DNA fragments comprising methylated adaptors; (b)contacting the tagmented DNA fragments with an array comprising aplurality of capture probes, wherein a capture probe in the plurality ofcapture probes comprises a spatial barcode, wherein if the capture probecomprises one or more cytosines, wherein the one or more cytosines aremethylated cytosines; (c) ligating the sequence comprising the tagmentedDNA fragments and the one or more methylated adaptors to the captureprobe, thereby creating a ligation product; (d) deaminating the ligationproduct; and (e) determining (i) all or a portion of the sequence of thespatial barcode or the complement thereof, and (ii) all or a portion ofthe sequence of the ligation product, or a complement thereof, and usingthe determined sequences of (i) and (ii) to identify the methylationstatus of the DNA, or a portion thereof, in the biological sample.

In some embodiments, the ligating comprises adding a splintoligonucleotide comprises (i) a sequence that binds specifically to aportion of the capture probe; and (ii) a sequence that bindsspecifically to a portion of the ligation product. In some embodiments,the splint oligonucleotide comprises a sequence that binds specificallyto a portion of the one or more methylated adaptors.

In some embodiments, the transposase enzyme is a Tn5 transposase enzyme,or a functional derivative thereof or a Tn7 transposase enzyme, or thefunctional derivative thereof.

In some embodiments, the one or more methylated adaptors comprises afirst methylated adaptor and a second methylated adaptor.

In some embodiments, the method described herein further comprisesincubating the ligation product with a terminal transferase and aplurality of deoxycytidine triphosphate (dCTP) molecules, therebyextending the ligation product at the 3′ end.

In some embodiments, the method described herein further comprisesamplifying the ligation product. In some embodiments, the methoddescribed herein further comprises amplifying the ligation product usinga primer sequence that is complementary to a poly-cysteine sequence atthe 3′ end of the ligation product.

In some embodiments, the ligating comprises enzymatic ligation orchemical ligation. In some embodiments, the enzymatic ligation utilizesa ligase. In some embodiments, the ligase is one or more of a splintRligase, a single stranded DNA ligase, or a T4 DNA ligase.

In some embodiments, the method described herein further comprises amigration step wherein the analyte migrates to the substrate. In someembodiments, the migration step is an active migration step comprisingapplying an electric field to the genomic DNA. In some embodiments, themigration step is a passive migration step.

In another aspect, described herein is a method of identifying themethylation status of a nucleic acid in a biological sample on a firstsubstrate, comprising: (a) deaminating the nucleic acid; (b) hybridizinga first probe and a second probe to the nucleic acid, wherein: the firstprobe comprises (i) a sequence complementary to at least a firstsequence of the nucleic acid and (ii) a sequence complementary to acapture domain of a capture probe on an array; and the second probecomprises a sequence complementary to at least a second sequence of thenucleic acid; (c) ligating the first probe and the second probe togenerate a ligation product; (d) aligning the first substrate with thesecond substrate comprising the array, such that at least a portion ofthe biological sample is aligned with at least a portion of the array,wherein the array comprises a plurality of capture probes, wherein acapture probe of the plurality of capture probes comprises: (i) aspatial barcode and (ii) the capture domain; (e) when the biologicalsample is aligned with at least a portion of the array, (i) releasingthe ligation product from the analyte and (ii) migrating the ligationproduct from the biological sample to the array; and (f) capturing theligation product with the capture domain.

In some embodiments, the method described herein further comprisesdetermining (i) all or a portion of the sequence of the spatial barcodeor the complement thereof and (ii) all or a portion of the sequence ofthe ligation product, or a complement thereof.

In some embodiments, the method described herein further comprises usingthe determined sequences of (i) and (ii) to identify the methylationstatus of the nucleic acid in the biological sample.

In some embodiments, the first probe is at least 75%, at least 80%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% complementary to the nucleic acid. In someembodiments, the first probe is about 15 to about 120 nucleotides inlength. In some embodiments, the first sequence is about 10 to about 60nucleotides in length.

In some embodiments, the second probe is at least 75%, at least 80%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% complementary to the nucleic acid. In someembodiments, the second probe is about 15 to about 120 nucleotides inlength. In some embodiments, the second sequence is about 10 to about 60nucleotides in length.

In some embodiments, the first sequence and the second sequence areadjacent sequences.

In some embodiments, at least one deaminated nucleotide is locatedbetween the first sequence and the second sequence. In some embodiments,at least two, at least three, at least four, at least five, or moredeaminated nucleotide is located between the first sequence and thesecond sequence.

In some embodiments, the method described herein further comprisesgenerating an extended first probe and/or an extended second probe usinga polymerase, wherein the extended first probe or the extended secondprobe comprises a sequence complementary to a sequence between the firstsequence and the second sequence.

In some embodiments, at least one deaminated nucleotide is located inthe first sequence. In some embodiments, the at least one deaminatednucleotide is located in the second sequence.

In some embodiments, the releasing comprises heating the biologicalsample. In some embodiments, step (e) further comprises contacting thebiological sample with a reagent medium comprising a permeabilizationreagent, optionally wherein the permeabilization reagent comprises aprotease.

In some embodiments, the ligating the first probe and the second probecomprises ligating via a ligase the first probe and the second probe. Insome embodiments, the ligating the first probe and the second probecomprises ligating via a ligase: (a) the first probe and the extendedsecond probe; or (b) the extended first probe and the second probe. Insome embodiments, the ligase is selected from a splintR ligase, a singlestranded DNA ligase, or a T4 DNA ligase.

In some embodiments, the first probe and the second probe are on acontiguous nucleic acid. In some embodiments, the ligating utilizes asplint oligonucleotide. In some embodiments, the splint oligonucleotidecomprises a first splint sequence that is substantially complementary tothe first probe and a second splint sequence that is substantiallycomplementary to the second probe.

In some embodiments, the capturing the ligation product compriseshybridizing the sequence complementary to a capture domain to thecapture domain. In some embodiments, the capturing the ligation productcomprises ligating the ligation product to the capture probe.

In some embodiments, the method described herein further comprisesextending the capture probe using the ligation product as a template;thereby generating an extended capture probe.

In another aspect, described herein is a method of identifying amethylation status of a nucleic acid in a biological sample on a firstsubstrate, the method comprising: (a) ligating a methylated adaptor to anucleic acid, generating an adapted nucleic acid fragment; (b)deaminating the adapted nucleic acid fragment; (c) aligning the firstsubstrate with a second substrate comprising an array, such that atleast a portion of the biological sample is aligned with at least aportion of the array, wherein the array comprises a plurality of captureprobes, wherein a capture probe of the plurality of capture probescomprises: (i) a spatial barcode and (ii) a capture domain; (d) when thebiological sample is aligned with at least a portion of the array,migrating the adapted nucleic acid fragment from the biological sampleto the array; and (e) capturing the adapted nucleic acid fragment withthe capture domain.

In some embodiments, the method described herein further comprisesdetermining (i) all or a portion of the sequence of the spatial barcodeor the complement thereof, and (ii) all or a portion of the sequence ofthe ligation product, or a complement thereof.

In some embodiments, the method described herein further comprises usingthe determined sequences of (i) and (ii) to identify the methylationstatus of the nucleic acid, or a portion thereof, in the biologicalsample.

In some embodiments, the methylated adaptor is ligated to the nucleicacid by tagmentation. In some embodiments, the methylated adaptor isligated to the nucleic acid at a 5′ end of the nucleic acid. In someembodiments, the methylated adaptor is ligated to the nucleic acid at a3′ end of the nucleic acid.

In some embodiments, the methylated adaptor is at least about 10nucleotides to about 50 nucleotides long.

In some embodiments, the methylated adaptor comprises at least one, atleast two, at least three, at least four, at least five, or moremethylated cytosines.

In some embodiments, the spatial barcode comprises at least one, atleast two, at least three, at least four, at least five, or moremethylated cytosines.

In some embodiments, the ligating the methylated adaptor to the nucleicacid comprises attaching the one or more methylated adaptors to the 5′and/or 3′ end of the nucleic acid using a transposase enzyme complexedwith a transposon, wherein the transposase enzyme is a Tn5 transposaseenzyme, or the functional derivative thereof.

In some embodiments, the capturing the adapted nucleic acid fragmentcomprises hybridizing the adapted nucleic acid fragment with the capturedomain. In some embodiments, the capturing the adapted nucleic acidfragment comprises ligating the adapted nucleic acid fragment with thecapture domain, wherein the ligating comprises enzymatic ligation orchemical ligation.

In some embodiments, the ligating utilizes a splint oligonucleotide,optionally wherein the splint oligonucleotide comprises a first splintsequence that is substantially complementary to the adapted nucleic acidfragment and a second splint sequence that is substantiallycomplementary to the capture probe.

In some embodiments, the method described herein further comprisesextending the capture probe using the adapted nucleic acid fragment as atemplate; thereby generating an extended capture probe.

In some embodiments, the method described herein further comprisesincubating the adapted nucleic acid fragment with a terminal transferaseand a plurality of deoxycytidine triphosphate (dCTP) molecules, therebygenerating an extended adapted nucleic acid fragment.

In some embodiments, the method described herein further comprisesamplifying the extended adapted nucleic acid fragment. In someembodiments, the method described herein further comprises amplifyingthe extended adapted nucleic acid fragment using a primer sequence thatis complementary to a poly-cysteine sequence at the 3′ end of theadapted nucleic acid fragment.

In some embodiments, the biological sample is a cancer tissue sample. Insome embodiments, the biological sample is from a subject treated with acancer therapy. In some embodiments, the nucleic acid is DNA. In someembodiments, the DNA is genomic DNA.

In some embodiments, the biological sample is a tissue sample,optionally wherein the tissue sample is a solid tissue sample,optionally wherein the tissue sample is a tissue section. In someembodiments, the biological sample is a fixed sample, a frozen sample, afresh sample, or a fresh frozen sample. In some embodiments, thebiological sample is a formalin fixed paraffin embedded (FFPE) sample.

In some embodiments, the first substrate is a slide. In someembodiments, the first substrate is a glass slide. In some embodiments,the first substrate does not comprise an array of capture probes.

In some embodiments, the deaminating comprises contacting the biologicalsample with a composition comprising sodium bisulfite. In someembodiments, the deaminating comprises treating the biological samplewith an enzyme. In some embodiments, the enzyme is a cytidine deaminaseor a demethylase.

In some embodiments, the second substrate is a glass slide. In someembodiments, a 5′ end of the capture probe is attached to the secondsubstrate.

In some embodiments, the array is a bead array. In some embodiments, a5′ end of the capture probe is attached to a bead of the bead array.

In some embodiments, the capture probe further comprises a uniquemolecular identifier (UMI).

In some embodiments, the capture probe further comprises one or morefunctional domains, a unique molecular identifier, a cleavage domain,and combinations thereof.

In some embodiments, the migrating is active. In some embodiments, themigrating is passive.

In some embodiments, the capture probe is extended using a polymerase.

In some embodiments, the determining comprises sequencing (i) all or apart of the sequence of the nucleic acid, or a complement thereof, and(ii) all or a part of the sequence of the spatial barcode, or acomplement thereof.

In some embodiments, the methylation status comprises identifying thatabout 1% to about 100% of cytosines comprises a methyl group.

In some embodiments, the method described herein further comprisesimaging the biological sample. In some embodiments, the imaging occursprior to deaminating the biological sample.

In another aspect, described herein is a kit comprising parts andinstruction for performing any one of the methods described herein.

In some embodiments, the kit comprises a substrate comprising aplurality of capture probes, wherein a capture probe of the plurality offirst capture probes comprises (i) a spatial barcode and (ii) a capturedomain.

In some embodiments, the kit comprises one or more enzyme. In someembodiments, the one or more enzyme is selected from the groupconsisting of a transposase, a ligase, a polymerase; a reversetranscriptase, a cytidine deaminase and a demethylase.

In some embodiments, the kit further comprises a transposome complexcomprising a transposase, a transposon sequence, and/or an adaptorsequence.

In another aspect, described herein is a composition comprising reagentsfor performing any one of the methods described herein.

In some embodiments, the composition comprises a substrate comprising aplurality of capture probes, wherein a capture probe of the plurality offirst capture probes comprises (i) a spatial barcode and (ii) a capturedomain.

In some embodiments, the composition comprises one or more enzyme. Insome embodiments, the one or more enzyme is selected from the groupconsisting of a transposase, a ligase, a polymerase; a reversetranscriptase, a cytidine deaminase and a demethylase.

In some embodiments, the composition further comprises a transposomecomplex comprising a transposase, a transposon sequence, and/or anadaptor sequence.

All publications, patents, patent applications, and informationavailable on the internet and mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication, patent, patent application, or item of information wasspecifically and individually indicated to be incorporated by reference.To the extent publications, patents, patent applications, and items ofinformation incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

Where values are described in terms of ranges, it should be understoodthat the description includes the disclosure of all possible sub-rangeswithin such ranges, as well as specific numerical values that fallwithin such ranges irrespective of whether a specific numerical value orspecific sub-range is expressly stated.

The term “each,” when used in reference to a collection of items, isintended to identify an individual item in the collection but does notnecessarily refer to every item in the collection, unless expresslystated otherwise, or unless the context of the usage clearly indicatesotherwise.

The singular form “a”, “an”, and “the” include plural references unlessthe context clearly dictates otherwise. For example, the term “a cell”includes one or more cells, comprising mixtures thereof “A and/or B” isused herein to include all of the following alternatives: “A”, “B”, “Aor B”, and “A and B”.

Various embodiments of the features of this disclosure are describedherein. However, it should be understood that such embodiments areprovided merely by way of example, and numerous variations, changes, andsubstitutions can occur to those skilled in the art without departingfrom the scope of this disclosure. It should also be understood thatvarious alternatives to the specific embodiments described herein arealso within the scope of this disclosure.

DESCRIPTION OF DRAWINGS

The following drawings illustrate certain embodiments of the featuresand advantages of this disclosure. These embodiments are not intended tolimit the scope of the appended claims in any manner. Like referencesymbols in the drawings indicate like elements.

FIG. 1 is a schematic diagram showing an example of a barcoded captureprobe, as described herein.

FIG. 2 is a schematic illustrating a cleavable capture probe, whereinthe cleaved capture probe can enter into a non-permeabilized cell andbind to target analytes within the sample.

FIG. 3 is a schematic diagram of an exemplary multiplexedspatially-barcoded feature.

FIG. 4 is a schematic showing the arrangement of barcoded featureswithin an array.

FIG. 5 is a schematic illustrating a side view of a diffusion-resistantmedium, e.g., a lid.

FIGS. 6A and 6B are schematics illustrating expanded FIG. 6A and sideviews FIG. 6B of an electrophoretic transfer system configured to directtranscript analytes toward a spatially-barcoded capture probe array.

FIG. 7 is a schematic illustrating an exemplary workflow protocolutilizing an electrophoretic transfer system.

FIG. 8A shows a schematic of an example analytical workflow in whichelectrophoretic migration of analytes is performed afterpermeabilization.

FIG. 8B shows a schematic of an example analytical workflow in whichelectrophoretic migration of analytes and permeabilization are performedsimultaneously.

FIG. 9A shows an example perpendicular, single slide configuration foruse during electrophoresis.

FIG. 9B shows an example parallel, single slide configuration for useduring electrophoresis.

FIG. 9C shows an example multi-slide configuration for use duringelectrophoresis.

FIG. 10 shows a schematic of an example analytical workflow ofgenerating a deaminated nucleic acid in a biological sample andidentifying a methylation status of a deaminated nucleic acid in abiological sample.

FIG. 11A shows a schematic of an example analytical workflow ofgenerating a deaminated nucleic acid in a biological sample

FIG. 11B shows a schematic of an example analytical workflow ofidentifying a methylation status of a deaminated nucleic acid in abiological sample.

FIG. 12 shows a schematic diagram depicting an exemplary two slidespatial array embodiment.

FIG. 13 shows a table providing median genes per spot and median uniquemolecular identifier (UMI) counts per spot in a formalin-fixed mousebrain sample.

FIG. 14 shows visual heat map results showing Log₁₀ UMI counts in acontrol single slide and a two slide spatial set up embodiments.

FIG. 15 shows spatial clustering analysis images in control andtwo-slide spatial array embodiments.

FIG. 16 shows a schematic of an example analytical workflow ofgenerating a deaminated nucleic acid in a biological sample.

FIG. 17 shows a schematic of an example analytical workflow ofgenerating a deaminated nucleic acid in a biological sample.

FIG. 18A shows a schematic of an example analytical workflow oftagmentation of DNA molecules in a biological sample and capture of thetagmented DNA fragments on spatial gene expression slides.

FIG. 18B shows a schematic of an example analytical workflow ofgenerating a library for whole genome methylation profiling (e.g., wholegenome bisulfite sequencing) using the captured tagmented DNA fragmentsfrom FIG. 18A.

FIG. 19A shows an exemplary embodiment of spatial deamination methodsdisclosed herein.

FIG. 19B shows the deaminated nucleic acid product after amplificationand before capture on an array.

FIGS. 20 and 21 show representative electropherogram of sequenced DNAmolecules after capture on an array.

FIG. 22 shows sequencing results for the captured and deaminated targetanalyte with representative percentages of adenine (A), cytosine (C),guanine (G), and thymine (T)

DETAILED DESCRIPTION

The Applicant has identified a need in the scientific community todetermine, spatially, the methylation status of a biological sample. Inparticular, due to heterogenous cell distribution in a biologicalsample, it is possible that the distribution of methylated nucleotides(e.g., methylated DNA) among cells in a biological sample can vary.Methylation of DNA is an important epigenetic modification. Themethylation status of each DNA molecule is variable and DNA methylationstatus in different cells is variable. This makes the analysis of DNAmethylation challenging and cumbersome. This is further confounded bythe fact that methylated-cytosine bases are not distinguishable fromunmethylated-cytosine bases in standard DNA sequencing technologies(e.g., sequencing-by synthesis or Sanger sequencing).

DNA methylation is a biological process by which methyl groups are addedto the DNA molecule, thereby changing gene activity without changing theunderlying DNA sequence. In mammals, epigenetic modifications such asDNA methylation occur at primarily cytosine/guanine (CG) dinucleotides.DNA methylation is typically found in promoter regions (known as CpGislands) and are associated with transcriptional repression. Forexample, a gene can be activated (e.g., “turned on”) in the presence ofopen chromatin and acetylated histones. In this instance, nucleotidesgenerally remain unmethylated. However, in the presence of a methylatednucleotide (e.g., a methylated cytosine), a chromosome can be condensed,resulting in de-activation of gene expression (e.g., expression is“turned off”). Thus, when located in a gene promoter, DNA methylationtypically acts to repress gene transcription. Two DNA bases, cytosineand adenine, can be methylated. Cytosine methylation is widespread inboth eukaryotes and prokaryotes. Methylation of cytosine to form5-methylcytosine occurs at the same 5′ position on the pyrimidine ringwhere the DNA base thymine's methyl group is located; the same positiondistinguishes thymine from the analogous RNA base uracil, which has nomethyl group. Spontaneous deamination of 5-methylcytosine converts it tothymine. This results in a T:G mismatch that can be identified throughsequencing techniques.

In recent decades, DNA methylation has been a subject of intense study,including how it occurs and where it occurs, and it has been discoveredthat methylation is an important component in numerous cellularprocesses, including embryonic development, genomic imprinting,X-chromosome inactivation, and preservation of chromosome stability. DNAmethylation is used as a differentiating marker in various settings,including cancer, neurology, some genetic diseases, development,cellular differentiation, model organism understanding, and duringtherapy (e.g., drug treatment).

Given the many processes in which methylation plays a part, errors inmethylation are also linked to a variety of devastating consequences,including several human diseases.

To study DNA methylation, many researchers rely on the use of adeaminating reagent, bisulfite: a chemical can be used in a processwhich converts unmethylated cytosines to thymines and leaves methylatedcytosines intact. However, the bisulfite is used under harsh conditionsthat fragment DNA, and are often not compatible with enzymes andreagents required for various applications. Herein are cost-effectiveand efficient techniques for determining a methylation status of abiological sample. The methods and compositions disclosed herein combinespatial analysis, multiple substrates, and methylation identificationtechniques.

Methods and Compositions for Spatial Analysis

Spatial analysis methodologies and compositions described herein canprovide a vast amount of analyte and/or expression data for a variety ofanalytes within a biological sample at high spatial resolution, whileretaining native spatial context. Spatial analysis methods andcompositions can include, e.g., the use of a capture probe including aspatial barcode (e.g., a nucleic acid sequence that provides informationas to the location or position of an analyte within a cell or a tissuesample (e.g., mammalian cell or a mammalian tissue sample) and a capturedomain that is capable of binding to an analyte (e.g., a protein and/ora nucleic acid) produced by and/or present in a cell. Spatial analysismethods and compositions can also include the use of a capture probehaving a capture domain that captures an intermediate agent for indirectdetection of an analyte. For example, the intermediate agent can includea nucleic acid sequence (e.g., a barcode) associated with theintermediate agent. Detection of the intermediate agent is thereforeindicative of the analyte in the cell or tissue sample.

Non-limiting aspects of spatial analysis methodologies and compositionsare described in U.S. Pat. Nos. 10,774,374, 10,724,078, 10,480,022,10,059,990, 10,041,949, 10,002,316, 9,879,313, 9,783,841, 9,727,810,9,593,365, 8,951,726, 8,604,182, 7,709,198, U.S. Patent ApplicationPublication Nos. 2020/239946, 2020/080136, 2020/0277663, 2020/024641,2019/330617, 2019/264268, 2020/256867, 2020/224244, 2019/194709,2019/161796, 2019/085383, 2019/055594, 2018/216161, 2018/051322,2018/0245142, 2017/241911, 2017/089811, 2017/067096, 2017/029875,2017/0016053, 2016/108458, 2015/000854, 2013/171621, WO 2018/091676, WO2020/176788, Rodriques et al., Science 363(6434):1463-1467, 2019; Lee etal., Nat. Protoc. 10(3):442-458, 2015; Trejo et al., PLoS ONE14(2):e0212031, 2019; Chen et al., Science 348(6233):aaa6090, 2015; Gaoet al., BMC Biol. 15:50, 2017; and Gupta et al., Nature Biotechnol.36:1197-1202, 2018; the Visium Spatial Gene Expression Reagent Kits UserGuide (e.g., Rev C, dated June 2020), and/or the Visium Spatial TissueOptimization Reagent Kits User Guide (e.g., Rev C, dated July 2020),both of which are available at the 10× Genomics Support Documentationwebsite, and can be used herein in any combination. Further non-limitingaspects of spatial analysis methodologies and compositions are describedherein.

Some general terminology that may be used in this disclosure can befound in Section (I)(b) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. Typically, a “barcode” is a label, oridentifier, that conveys or is capable of conveying information (e.g.,information about an analyte in a sample, a bead, and/or a captureprobe). A barcode can be part of an analyte, or independent of ananalyte. A barcode can be attached to an analyte. A particular barcodecan be unique relative to other barcodes. For the purpose of thisdisclosure, an “analyte” can include any biological substance,structure, moiety, or component to be analyzed. The term “target” cansimilarly refer to an analyte of interest.

Analytes can be broadly classified into one of two groups: nucleic acidanalytes, and non-nucleic acid analytes. Examples of non-nucleic acidanalytes include, but are not limited to, lipids, carbohydrates,peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins,phosphoproteins, specific phosphorylated or acetylated variants ofproteins, amidation variants of proteins, hydroxylation variants ofproteins, methylation variants of proteins, ubiquitylation variants ofproteins, sulfation variants of proteins, viral proteins (e.g., viralcapsid, viral envelope, viral coat, viral accessory, viralglycoproteins, viral spike, etc.), extracellular and intracellularproteins, antibodies, and antigen binding fragments. In someembodiments, the analyte(s) can be localized to subcellular location(s),including, for example, organelles, e.g., mitochondria, Golgi apparatus,endoplasmic reticulum, chloroplasts, endocytic vesicles, exocyticvesicles, vacuoles, lysosomes, etc. In some embodiments, analyte(s) canbe peptides or proteins, including without limitation antibodies andenzymes. Additional examples of analytes can be found in Section (I)(c)of WO 2020/176788 and/or U.S. Patent Application Publication No.2020/0277663. In some embodiments, an analyte can be detectedindirectly, such as through detection of an intermediate agent, forexample, a ligation product or an analyte capture agent (e.g., anoligonucleotide-conjugated antibody), such as those described herein.

A “biological sample” is typically obtained from the subject foranalysis using any of a variety of techniques including, but not limitedto, biopsy, surgery, and laser capture microscopy (LCM), and generallyincludes cells and/or other biological material from the subject. Insome embodiments, a biological sample can be a tissue section. In someembodiments, a biological sample can be a fixed and/or stainedbiological sample (e.g., a fixed and/or stained tissue section).Non-limiting examples of stains include histological stains (e.g.,hematoxylin and/or eosin) and immunological stains (e.g., fluorescentstains). In some embodiments, a biological sample (e.g., a fixed and/orstained biological sample) can be imaged. Biological samples are alsodescribed in Section (I)(d) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some embodiments, a biological sample is permeabilized with one ormore permeabilization reagents. For example, permeabilization of abiological sample can facilitate analyte capture. Exemplarypermeabilization agents and conditions are described in Section(I)(d)(ii)(13) or the Exemplary Embodiments Section of WO 2020/176788and/or U.S. Patent Application Publication No. 2020/0277663.

Array-based spatial analysis methods involve the transfer of one or moreanalytes from a biological sample to an array of features on asubstrate, where each feature is associated with a unique spatiallocation on the array. Subsequent analysis of the transferred analytesincludes determining the identity of the analytes and the spatiallocation of the analytes within the biological sample. The spatiallocation of an analyte within the biological sample is determined basedon the feature to which the analyte is bound (e.g., directly orindirectly) on the array, and the feature's relative spatial locationwithin the array.

A “capture probe” refers to any molecule capable of capturing (directlyor indirectly) and/or labelling an analyte (e.g., an analyte ofinterest) in a biological sample. In some embodiments, the capture probeis a nucleic acid or a polypeptide. In some embodiments, the captureprobe includes a barcode (e.g., a spatial barcode and/or a uniquemolecular identifier (UMI)) and a capture domain). In some embodiments,a capture probe can include a cleavage domain and/or a functional domain(e.g., a primer-binding site, such as for next-generation sequencing(NGS)). See, e.g., Section (II)(b) (e.g., subsections (i)-(vi)) of WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.Generation of capture probes can be achieved by any appropriate method,including those described in Section (II)(d)(ii) of WO 2020/176788and/or U.S. Patent Application Publication No. 2020/0277663.

FIG. 1 is a schematic diagram showing an exemplary capture probe, asdescribed herein. As shown, the capture probe 102 is optionally coupledto a feature 101 by a cleavage domain 103, such as a disulfide linker.The capture probe can include a functional sequence 104 that are usefulfor subsequent processing. The functional sequence 104 can include allor a part of sequencer specific flow cell attachment sequence (e.g., aP5 or P7 sequence), all or a part of a sequencing primer sequence,(e.g., a R1 primer binding site, a R2 primer binding site), orcombinations thereof. The capture probe can also include a spatialbarcode 105. The capture probe can also include a unique molecularidentifier (UMI) sequence 106. While FIG. 1 shows the spatial barcode105 as being located upstream (5′) of UMI sequence 106, it is to beunderstood that capture probes wherein UMI sequence 106 is locatedupstream (5′) of the spatial barcode 105 is also suitable for use in anyof the methods described herein. The capture probe can also include acapture domain 107 to facilitate capture of a target analyte.

The capture domain can have a sequence complementary to a sequence of anucleic acid analyte. The capture domain can have a sequencecomplementary to a ligation product described herein. The capture domaincan have a sequence complementary to a capture handle sequence presentin an analyte capture agent. The capture domain can have a sequencecomplementary to a splint oligonucleotide. Such splint oligonucleotide,in addition to having a sequence complementary to a capture domain of acapture probe, can have a sequence complementary to a sequence of anucleic acid analyte, a portion of a ligation product described herein,a capture handle sequence described herein, and/or a methylated adaptordescribed herein.

The functional sequences can generally be selected for compatibilitywith any of a variety of different sequencing systems, e.g., Ion TorrentProton or PGM, Illumina sequencing instruments, PacBio, Oxford Nanopore,etc., and the requirements thereof. In some embodiments, functionalsequences can be selected for compatibility with non-commercializedsequencing systems. Examples of such sequencing systems and techniques,for which suitable functional sequences can be used, include (but arenot limited to) Ion Torrent Proton or PGM sequencing, Illuminasequencing, PacBio SMRT sequencing, and Oxford Nanopore sequencing.Further, in some embodiments, functional sequences can be selected forcompatibility with other sequencing systems, includingnon-commercialized sequencing systems.

In some embodiments, the spatial barcode 105 and functional sequences104 is common to all of the probes attached to a given feature. In someembodiments, the UMI sequence 106 of a capture probe attached to a givenfeature is different from the UMI sequence of a different capture probeattached to the given feature.

FIG. 2 is a schematic illustrating a cleavable capture probe, whereinthe cleaved capture probe can enter into a non-permeabilized cell andbind to analytes within the sample. The capture probe 201 contains acleavage domain 202, a cell penetrating peptide 203, a reporter molecule204, and a disulfide bond (—S—S—). 205 represents all other parts of acapture probe, for example a spatial barcode and a capture domain.Cleavable capture probe are further described in WO 2020/176788 and/orU.S. Patent Application Publication No. 2020/0277663, each of which isincorporated by reference in its entirety.

For multiple capture probes that are attached to a common array feature,the one or more spatial barcode sequences of the multiple capture probescan include sequences that are the same for all capture probes coupledto the feature, and/or sequences that are different across all captureprobes coupled to the feature.

FIG. 3 is a schematic diagram of an exemplary multiplexedspatially-barcoded feature. In FIG. 3 , the feature 301 can be coupledto spatially-barcoded capture probes, wherein the spatially-barcodedprobes of a particular feature can possess the same spatial barcode, buthave different capture domains designed to associate the spatial barcodeof the feature with more than one target analyte. For example, a featuremay be coupled to four different types of spatially-barcoded captureprobes, each type of spatially-barcoded capture probe possessing thespatial barcode 302. One type of capture probe associated with thefeature includes the spatial barcode 302 in combination with a poly(T)capture domain 303, designed to capture mRNA target analytes. A secondtype of capture probe associated with the feature includes the spatialbarcode 302 in combination with a random N-mer capture domain 304 forgDNA analysis. A third type of capture probe associated with the featureincludes the spatial barcode 302 in combination with a capture domaincomplementary to a capture handle sequence of an analyte capture agentof interest 305. A fourth type of capture probe associated with thefeature includes the spatial barcode 302 in combination with a capturedomain that can specifically bind a nucleic acid molecule 306 that canfunction in a CRISPR assay (e.g., CRISPR/Cas9). While only fourdifferent capture probe-barcoded constructs are shown in FIG. 3 ,capture-probe barcoded constructs can be tailored for analyses of anygiven analyte associated with a nucleic acid and capable of binding withsuch a construct. For example, the schemes shown in FIG. 3 can also beused for concurrent analysis of other analytes disclosed herein,including, but not limited to: (a) mRNA, a lineage tracing construct,cell surface or intracellular proteins and metabolites, and gDNA; (b)mRNA, accessible chromatin (e.g., ATAC-seq, DNase-seq, and/or MNase-seq)cell surface or intracellular proteins and metabolites, and aperturbation agent (e.g., a CRISPR crRNA/sgRNA, TALEN, zinc fingernuclease, and/or antisense oligonucleotide as described herein); (c)mRNA, cell surface or intracellular proteins and/or metabolites, abarcoded labelling agent (e.g., the MHC multimers described herein), anda V(D)J sequence of an immune cell receptor (e.g., T-cell receptor). Insome embodiments, a perturbation agent can be a small molecule, anantibody, a drug, an aptamer, a miRNA, a physical environmental (e.g.,temperature change), or any other known perturbation agents. See, e.g.,Section (II)(b) (e.g., subsections (i)-(vi)) of WO 2020/176788 and/orU.S. Patent Application Publication No. 2020/0277663. Generation ofcapture probes can be achieved by any appropriate method, includingthose described in Section (II)(d)(ii) of WO 2020/176788 and/or U.S.Patent Application Publication No. 2020/0277663.

Capture probes attached to a single array feature can include identical(or common) spatial barcode sequences, different spatial barcodesequences, or a combination of both. Capture probes attached to afeature can include multiple sets of capture probes. Capture probes of agiven set can include identical spatial barcode sequences. The identicalspatial barcode sequences can be different from spatial barcodesequences of capture probes of another set.

The plurality of capture probes can include spatial barcode sequences(e.g., nucleic acid barcode sequences) that are associated with specificlocations on a spatial array. For example, a first plurality of captureprobes can be associated with a first region, based on a spatial barcodesequence common to the capture probes within the first region, and asecond plurality of capture probes can be associated with a secondregion, based on a spatial barcode sequence common to the capture probeswithin the second region. The second region may or may not be associatedwith the first region. Additional pluralities of capture probes can beassociated with spatial barcode sequences common to the capture probeswithin other regions. In some embodiments, the spatial barcode sequencescan be the same across a plurality of capture probe molecules.

In some embodiments, multiple different spatial barcodes areincorporated into a single arrayed capture probe. For example, a mixedbut known set of spatial barcode sequences can provide a strongeraddress or attribution of the spatial barcodes to a given spot orlocation, by providing duplicate or independent confirmation of theidentity of the location. In some embodiments, the multiple spatialbarcodes represent increasing specificity of the location of theparticular array point.

In some embodiments, more than one analyte type (e.g., nucleic acids andproteins) from a biological sample can be detected (e.g., simultaneouslyor sequentially) using any appropriate multiplexing technique, such asthose described in Section (IV) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some embodiments, detection of one or more analytes (e.g., proteinanalytes) can be performed using one or more analyte capture agents. Asused herein, an “analyte capture agent” refers to an agent thatinteracts with an analyte (e.g., an analyte in a biological sample) andwith a capture probe (e.g., a capture probe attached to a substrate or afeature) to identify the analyte. In some embodiments, the analytecapture agent includes: (i) an analyte binding moiety (e.g., that bindsto an analyte), for example, an antibody or antigen-binding fragmentthereof; (ii) analyte binding moiety barcode; and (iii) an analytecapture sequence or capture handle sequence. As used herein, the term“analyte binding moiety barcode” refers to a barcode that is associatedwith or otherwise identifies the analyte binding moiety. As used herein,the term “analyte capture sequence” or “capture handle sequence” refersto a region or moiety configured to hybridize to, bind to, couple to, orotherwise interact with a capture domain of a capture probe. In someembodiments, a capture handle sequence is complementary to a capturedomain of a capture probe. In some cases, an analyte binding moietybarcode (or portion thereof) may be able to be removed (e.g., cleaved)from the analyte capture agent. Additional description of analytecapture agents can be found in Section (II)(b)(ix) of WO 2020/176788and/or Section (II)(b)(viii) U.S. Patent Application Publication No.2020/0277663.

There are at least two methods to associate a spatial barcode with oneor more neighboring cells, such that the spatial barcode identifies theone or more cells, and/or contents of the one or more cells, asassociated with a particular spatial location. One method is to promoteanalytes or analyte proxies (e.g., intermediate agents) out of a celland towards a spatially-barcoded array (e.g., includingspatially-barcoded capture probes). Another method is to cleavespatially-barcoded capture probes from an array and promote thespatially-barcoded capture probes towards and/or into or onto thebiological sample.

In some cases, capture probes may be configured to prime, replicate, andconsequently yield optionally barcoded extension products from atemplate (e.g., a DNA template, such as an analyte or an intermediateagent (e.g., a ligation product or an analyte capture agent), or aportion thereof), or derivatives thereof (see, e.g., Section(II)(b)(vii) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663 regarding extended capture probes). In somecases, capture probes may be configured to form ligation products with atemplate (e.g., a DNA template, such as an analyte or an intermediateagent, or portion thereof), thereby creating ligations products thatserve as proxies for a template.

As used herein, an “extended capture probe” refers to a capture probehaving additional nucleotides added to the terminus (e.g., 3′ or 5′ end)of the capture probe thereby extending the overall length of the captureprobe. For example, an “extended 3′ end” indicates additionalnucleotides were added to the most 3′ nucleotide of the capture probe toextend the length of the capture probe, for example, by polymerizationreactions used to extend nucleic acid molecules including templatedpolymerization catalyzed by a polymerase (e.g., a DNA polymerase or areverse transcriptase). In some embodiments, extending the capture probeincludes adding to a 3′ end of a capture probe a nucleic acid sequencethat is complementary to a nucleic acid sequence of an analyte orintermediate agent specifically bound to the capture domain of thecapture probe. In some embodiments, the capture probe is extended usingreverse transcription. In some embodiments, the capture probe isextended using one or more DNA polymerases. The extended capture probesinclude the sequence of the capture probe and the sequence of thespatial barcode of the capture probe.

In some embodiments, extended capture probes are amplified (e.g., inbulk solution or on the array) to yield quantities that are sufficientfor downstream analysis, e.g., via DNA sequencing. In some embodiments,extended capture probes (e.g., DNA molecules) act as templates for anamplification reaction (e.g., a polymerase chain reaction).

Additional variants of spatial analysis methods, including in someembodiments, an imaging step, are described in Section (II)(a) of WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.Analysis of captured analytes (and/or intermediate agents or portionsthereof), for example, including sample removal, extension of captureprobes, sequencing (e.g., of a cleaved extended capture probe and/or acDNA molecule complementary to an extended capture probe), sequencing onthe array (e.g., using, for example, in situ hybridization or in situligation approaches), temporal analysis, and/or proximity capture, isdescribed in Section (II)(g) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663. Some quality control measuresare described in Section (II)(h) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

Spatial information can provide information of biological and/or medicalimportance. For example, the methods and compositions described hereincan allow for: identification of one or more biomarkers (e.g.,diagnostic, prognostic, and/or for determination of efficacy of atreatment) of a disease or disorder; identification of a candidate drugtarget for treatment of a disease or disorder; identification (e.g.,diagnosis) of a subject as having a disease or disorder; identificationof stage and/or prognosis of a disease or disorder in a subject;identification of a subject as having an increased likelihood ofdeveloping a disease or disorder; monitoring of progression of a diseaseor disorder in a subject; determination of efficacy of a treatment of adisease or disorder in a subject; identification of a patientsubpopulation for which a treatment is effective for a disease ordisorder; modification of a treatment of a subject with a disease ordisorder; selection of a subject for participation in a clinical trial;and/or selection of a treatment for a subject with a disease ordisorder.

Spatial information can provide information of biological importance.For example, the methods and compositions described herein can allowfor: identification of transcriptome and/or proteome expression profiles(e.g., in healthy and/or diseased tissue); identification of multipleanalyte types in close proximity (e.g., nearest neighbor analysis);determination of up- and/or down-regulated genes and/or proteins indiseased tissue; characterization of tumor microenvironments;characterization of tumor immune responses; characterization of cellstypes and their co-localization in tissue; and identification of geneticvariants within tissues (e.g., based on gene and/or protein expressionprofiles associated with specific disease or disorder biomarkers).

Typically, for spatial array-based methods, a substrate functions as asupport for direct or indirect attachment of capture probes to featuresof the array. A “feature” is an entity that acts as a support orrepository for various molecular entities used in spatial analysis. Insome embodiments, some or all of the features in an array arefunctionalized for analyte capture. Exemplary substrates are describedin Section (II)(c) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. Exemplary features and geometricattributes of an array can be found in Sections (II)(d)(i),(II)(d)(iii), and (II)(d)(iv) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

FIG. 4 depicts an exemplary arrangement of barcoded features within anarray. From left to right, FIG. 4 shows (L) a slide including sixspatially-barcoded arrays, (C) an enlarged schematic of one of the sixspatially-barcoded arrays, showing a grid of barcoded features inrelation to a biological sample, and (R) an enlarged schematic of onesection of an array, showing the specific identification of multiplefeatures within the array (labelled as ID578, ID579, ID560, etc.).

Generally, analytes and/or intermediate agents (or portions thereof) canbe captured when contacting a biological sample with a substrateincluding capture probes (e.g., a substrate with capture probesembedded, spotted, printed, fabricated on the substrate, or a substratewith features (e.g., beads, wells) comprising capture probes). As usedherein, “contact,” “contacted,” and/or “contacting,” a biological samplewith a substrate refers to any contact (e.g., direct or indirect) suchthat capture probes can interact (e.g., bind covalently ornon-covalently (e.g., hybridize)) with analytes from the biologicalsample. Capture can be achieved actively (e.g., using electrophoresis)or passively (e.g., using diffusion). Analyte capture is furtherdescribed in Section (II)(e) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

FIG. 5 is an illustration of an exemplary use of a diffusion-resistantmedium. A diffusion-resistant medium/lid 502 can be contacted with asample 503. In FIG. 5 , a glass slide 504 is populated withspatially-barcoded capture probes 506, and the sample 503, 505 iscontacted with the array 504, 506. A diffusion-resistant medium/lid 502can be applied to the sample 503, wherein the sample 503 is disposedbetween a diffusion-resistant medium 502 and a capture probe coatedslide 504. When a permeabilization solution 501 is applied to thesample, the diffusion-resistant medium/lid 502 directs the migration ofthe analytes 505 toward proximal capture probes 506 by reducingdiffusion of the analytes out into the medium. Alternatively, thediffusion resistant medium/lid may contain permeabilization reagents.

In some cases, spatial analysis can be performed by attaching and/orintroducing a molecule (e.g., a peptide, a lipid, or a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) to a biologicalsample (e.g., to a cell in a biological sample). In some embodiments, aplurality of molecules (e.g., a plurality of nucleic acid molecules)having a plurality of barcodes (e.g., a plurality of spatial barcodes)are introduced to a biological sample (e.g., to a plurality of cells ina biological sample) for use in spatial analysis. In some embodiments,after attaching and/or introducing a molecule having a barcode to abiological sample, the biological sample can be physically separated(e.g., dissociated) into single cells or cell groups for analysis. Somesuch methods of spatial analysis are described in Section (III) of WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

In some cases, spatial analysis can be performed by detecting multipleoligonucleotides that hybridize to an analyte. In some instances, forexample, spatial analysis can be performed using RNA-templated ligation(RTL). Methods of RTL have been described previously. See, e.g., Credleet al., Nucleic Acids Res. 2017 Aug. 21; 45(14):e128. Typically, RTLincludes hybridization of two oligonucleotides to adjacent sequences onan analyte (e.g., an RNA molecule, such as an mRNA molecule). In someinstances, the oligonucleotides are DNA molecules. In some instances,one of the oligonucleotides includes at least two ribonucleic acid basesat the 3′ end and/or the other oligonucleotide includes a phosphorylatednucleotide at the 5′ end. In some instances, one of the twooligonucleotides includes a capture domain (e.g., a poly(A) sequence, anon-homopolymeric sequence). After hybridization to the analyte, aligase (e.g., SplintR ligase) ligates the two oligonucleotides together,creating a ligation product. In some instances, the two oligonucleotideshybridize to sequences that are not adjacent to one another. Forexample, hybridization of the two oligonucleotides creates a gap betweenthe hybridized oligonucleotides. In some instances, a polymerase (e.g.,a DNA polymerase) can extend one of the oligonucleotides prior toligation. After ligation, the ligation product is released from theanalyte. In some instances, the ligation product is released using anendonuclease (e.g., RNAse H). The released ligation product can then becaptured by capture probes (e.g., instead of direct capture of ananalyte) on an array, optionally amplified, and sequenced, thusdetermining the location and optionally the abundance of the analyte inthe biological sample.

During analysis of spatial information, sequence information for aspatial barcode associated with an analyte is obtained, and the sequenceinformation can be used to provide information about the spatialdistribution of the analyte in the biological sample. Various methodscan be used to obtain the spatial information. In some embodiments,specific capture probes and the analytes they capture are associatedwith specific locations in an array of features on a substrate. Forexample, specific spatial barcodes can be associated with specific arraylocations prior to array fabrication, and the sequences of the spatialbarcodes can be stored (e.g., in a database) along with specific arraylocation information, so that each spatial barcode uniquely maps to aparticular array location.

Alternatively, specific spatial barcodes can be deposited atpredetermined locations in an array of features during fabrication suchthat at each location, only one type of spatial barcode is present sothat spatial barcodes are uniquely associated with a single feature ofthe array. Where necessary, the arrays can be decoded using any of themethods described herein so that spatial barcodes are uniquelyassociated with array feature locations, and this mapping can be storedas described above.

When sequence information is obtained for capture probes and/or analytesduring analysis of spatial information, the locations of the captureprobes and/or analytes can be determined by referring to the storedinformation that uniquely associates each spatial barcode with an arrayfeature location. In this manner, specific capture probes and capturedanalytes are associated with specific locations in the array offeatures. Each array feature location represents a position relative toa coordinate reference point (e.g., an array location, a fiducialmarker) for the array. Accordingly, each feature location has an“address” or location in the coordinate space of the array.

Some exemplary spatial analysis workflows are described in the ExemplaryEmbodiments section of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. See, for example, the Exemplary embodimentstarting with “In some non-limiting examples of the workflows describedherein, the sample can be immersed . . . ” of WO 2020/176788 and/or U.S.Patent Application Publication No. 2020/0277663. See also, e.g., theVisium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev C,dated June 2020), and/or the Visium Spatial Tissue Optimization ReagentKits User Guide (e.g., Rev C, dated July 2020).

In some embodiments, spatial analysis can be performed using dedicatedhardware and/or software, such as any of the systems described inSections (II)(e)(ii) and/or (V) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663, or any of one or more of thedevices or methods described in Sections Control Slide for Imaging,Methods of Using Control Slides and Substrates for, Systems of UsingControl Slides and Substrates for Imaging, and/or Sample and ArrayAlignment Devices and Methods, Informational labels of WO 2020/123320.

Suitable systems for performing spatial analysis can include componentssuch as a chamber (e.g., a flow cell or sealable, fluid-tight chamber)for containing a biological sample. The biological sample can be mountedfor example, in a biological sample holder. One or more fluid chamberscan be connected to the chamber and/or the sample holder via fluidconduits, and fluids can be delivered into the chamber and/or sampleholder via fluidic pumps, vacuum sources, or other devices coupled tothe fluid conduits that create a pressure gradient to drive fluid flow.One or more valves can also be connected to fluid conduits to regulatethe flow of reagents from reservoirs to the chamber and/or sampleholder.

The systems can optionally include a control unit that includes one ormore electronic processors, an input interface, an output interface(such as a display), and a storage unit (e.g., a solid state storagemedium such as, but not limited to, a magnetic, optical, or other solidstate, persistent, writeable and/or re-writeable storage medium). Thecontrol unit can optionally be connected to one or more remote devicesvia a network. The control unit (and components thereof) can generallyperform any of the steps and functions described herein. Where thesystem is connected to a remote device, the remote device (or devices)can perform any of the steps or features described herein. The systemscan optionally include one or more detectors (e.g., CCD, CMOS) used tocapture images. The systems can also optionally include one or morelight sources (e.g., LED-based, diode-based, lasers) for illuminating asample, a substrate with features, analytes from a biological samplecaptured on a substrate, and various control and calibration media.

The systems can optionally include software instructions encoded and/orimplemented in one or more of tangible storage media and hardwarecomponents such as application specific integrated circuits. Thesoftware instructions, when executed by a control unit (and inparticular, an electronic processor) or an integrated circuit, can causethe control unit, integrated circuit, or other component executing thesoftware instructions to perform any of the method steps or functionsdescribed herein.

In some cases, the systems described herein can detect (e.g., registeran image) the biological sample on the array. Exemplary methods todetect the biological sample on an array are described in PCTApplication No. 2020/061064 and/or U.S. patent application Ser. No.16/951,854.

Prior to transferring analytes from the biological sample to the arrayof features on the substrate, the biological sample can be aligned withthe array. Alignment of a biological sample and an array of featuresincluding capture probes can facilitate spatial analysis, which can beused to detect differences in analyte presence and/or level withindifferent positions in the biological sample, for example, to generate athree-dimensional map of the analyte presence and/or level. Exemplarymethods to generate a two- and/or three-dimensional map of the analytepresence and/or level are described in PCT Application No. 2020/053655and spatial analysis methods are generally described in WO 2020/061108and/or U.S. patent application Ser. No. 16/951,864.

In some cases, a map of analyte presence and/or level can be aligned toan image of a biological sample using one or more fiducial markers,e.g., objects placed in the field of view of an imaging system whichappear in the image produced, as described in the Substrate AttributesSection, Control Slide for Imaging Section of WO 2020/123320, PCTApplication No. 2020/061066, and/or U.S. patent application Ser. No.16/951,843. Fiducial markers can be used as a point of reference ormeasurement scale for alignment (e.g., to align a sample and an array,to align two substrates, to determine a location of a sample or array ona substrate relative to a fiducial marker) and/or for quantitativemeasurements of sizes and/or distance.

Determination of Methylation Status in a Nucleic Acid

Disclosed herein are methods and compositions for determining themethylation status of a biological sample. Methylation status can bedetermined using methods known in the art (e.g., deamination of anucleic acid). The methods disclosed herein combine methylation statuswith spatial analysis to determine the location of a methylated nucleicacid (e.g., a DNA molecule) at a spatial location in a sample.Furthermore, the methods disclosed herein allow spatial profiling ofmethylation status of a biological sample placed on a standardhistological substrate, e.g., a standard slide.

In exemplary methods disclosed herein, a biological sample is providedon a first substrate, which can be any slide (e.g., a glass slide), anda deamination step can be performed in situ on the glass slide. Probes(e.g., a first probe, a second probe, or a methylated adaptor) interactwith a nucleic acid of the biological sample. The biological sample ispermeabilized and a probe-containing nucleic acid migrates to a secondsubstrate, which includes an array having a plurality of capture probes.The sequence and location of the probe-containing nucleic acid can bedetermined, and based on the sequence, the methylation status at aparticular location can be determined.

Methods for Identifying Methylation Status of an Analyte in a BiologicalSample Provided herein are methods for identifying a methylation statusof an analyte in a biological sample. “Methylation status” as usedherein refers to identifying the presence or absence of one or moremethyl groups in an analyte. In some instances, the one or more methylgroups is on one or more cytosines. In some instances, the disclosurefeatures a method for identifying a methylation status of an analyte ina biological sample, the method comprising: (a) contacting thebiological sample with an array comprising a plurality of captureprobes, wherein a capture probe in the plurality of capture probescomprises (i) a spatial barcode and (ii) a capture domain comprising atleast one methylated cytosine; (b) deaminating the analyte; (c)contacting the analyte with a plurality of probes comprising a firstprobe and a second probe, wherein the first probe comprises (i) asequence complementary to at least a first sequence of the analyte and(ii) a sequence complementary to the capture domain; and the secondprobe comprises a sequence complementary to at least a second sequenceof the analyte; (d) ligating the first probe and the second probe,thereby generating a ligation product; (e) hybridizing the ligationproduct to the capture probe; (f) extending the capture probe using theligation product as a template; thereby generating an extended captureprobe; (g) amplifying the extended capture probe to produce a pluralityof nucleic acids; and (h) determining (i) all or a portion of thesequence of the spatial barcode or the complement thereof, and (ii) allor a portion of the sequence of the analyte, or a complement thereof,and using the determined sequences of (i) and (ii) to identify themethylation status of the analyte in the biological sample.

In another feature, disclosed herein is a method comprising: (a)contacting the biological sample with an array, wherein the arraycomprises a plurality of capture probes, wherein a capture probe in theplurality of capture probes comprises (i) a spatial barcode and (ii) acapture domain; (b) deaminating the analyte; (c) contacting the analytewith a plurality of probes, wherein a probe in the plurality of probescomprises (i) a binding moiety that binds specifically to at least aportion of the analyte and (ii) an overhang sequence; (d) extending theprobe using the analyte as a template, thereby generating an extendedprobe; (e) hybridizing the extended probe to the capture probe; (f)extending the capture probe using the extended probe as a template,thereby generating an extended capture probe; (g) amplifying theextended capture probe to produce a plurality of nucleic acids; and (h)determining (i) all or a portion of the sequence of the spatial barcodeor the complement thereof, and (ii) all or a portion of the sequence ofthe analyte, or a complement thereof, and using the determined sequencesof (i) and (ii) to identify the methylation status of the analyte in thebiological sample.

Also provided herein are methods for identifying a methylation status ofan analyte in a biological sample, the method comprising: (a) contactingthe biological sample with an array, wherein the array comprises aplurality of capture probes, wherein a capture probe in the plurality ofcapture probes comprises (i) a spatial barcode and (ii) a capturedomain; wherein the capture domain comprises at least one methylatedcytosine; (b) deaminating the analyte; (c) contacting the analyte with aplurality of probes comprising a first probe and a second probe, whereinthe first probe comprises (i) a first binding moiety that bindsspecifically to at least a portion of the analyte and (ii) a firstoverhang sequence; and the second probe comprises (i) a second bindingmoiety that binds specifically to at least a portion of the analyte thatis adjacent to the portion of the analyte that is bound to the firstbinding moiety and (ii) a second overhang sequence; and (d) extendingthe first probe using the analyte as a template; (e) ligating the firstprobe and the second probe, thereby generating an extended probe; (f)hybridizing the extended probe to the capture probe; (g) extending thecapture probe using the extended probe as a template; thereby generatingan extended capture probe; (h) amplifying the extended capture probe toproduce a plurality of nucleic acids; and (i) determining (i) all or aportion of the sequence of the spatial barcode or the complementthereof, and (ii) all or a portion of the sequence of the analyte, or acomplement thereof, and using the determined sequences of (i) and (ii)to identify the methylation status of the analyte in the biologicalsample. In some embodiments, ligating comprises enzymatic ligation orchemical ligation. In some embodiments, the enzymatic ligation utilizesa ligase.

Also provided herein are methods for identifying a methylation status ofan analyte, e.g., a nucleic acid, in a biological sample. “Methylationstatus” as used herein refers to identifying the presence or absence ofone or more methyl groups in an analyte (e.g., on a cytosine). In someinstances, methylation status can be an absolute number of methylatedcytosines or non-methylated cytosines in a nucleic acid. In someinstances, methylation status can be a percentage of cytosines that areeither methylated or non-methylated in a nucleic acid.

In some instances, the disclosure features a method for identifying amethylation status of a nucleic acid in a biological sample. In someinstances, the biological sample is placed on a substrate that does notinclude capture probes. In this way, all steps of deamination arecarried out on this substrate. The methods provided herein allows thedeamination steps to be separated from the spatial analysis of theanalyte, e.g., nucleic acid. In an exemplary embodiment, the biologicalsample is placed on a regular slide, e.g., a glass slide that does nothave capture probes. Because the deamination is separated from thecapturing of molecules on the spatial array, the capture probes of thespatial array is not affected by the deamination reagents.

In some exemplary methods, provided herein are methods of identifyingthe methylation status of a nucleic acid in a biological sample on afirst substrate. In some instances, the methods include (a) deaminatingthe nucleic acid; (b) hybridizing a first probe and a second probe tothe nucleic acid, wherein: the first probe comprises (i) a sequencecomplementary to at least a first sequence of the nucleic acid and (ii)a sequence complementary to a capture domain of a capture probe on anarray; and the second probe comprises a sequence complementary to atleast a second sequence of the nucleic acid; (c) ligating the firstprobe and the second probe to generate a ligation product; (d) aligningthe first substrate with the second substrate comprising the array, suchthat at least a portion of the biological sample is aligned with atleast a portion of the array, wherein the array comprises a plurality ofcapture probes, wherein a capture probe of the plurality of captureprobes comprises: (i) a spatial barcode and (ii) the capture domain; (e)when the biological sample is aligned with at least a portion of thearray, (i) releasing the ligation product from the analyte and (ii)migrating the ligation product from the biological sample to the array;and (f) capturing the ligation product with the capture domain.

In other exemplary methods, provided herein are methods of identifying amethylation status of a nucleic acid in a biological sample on a firstsubstrate. In some instances, the methods include (a) ligating amethylated adaptor to a nucleic acid, generating an adapted (e.g.,tagmented) nucleic acid fragment; (b) deaminating the adapted (e.g.,tagmented) nucleic acid fragment; (c) aligning the first substrate witha second substrate comprising an array, such that at least a portion ofthe biological sample is aligned with at least a portion of the array,wherein the array comprises a plurality of capture probes, wherein acapture probe of the plurality of capture probes comprises: (i) aspatial barcode and (ii) a capture domain; (d) when the biologicalsample is aligned with at least a portion of the array, migrating theadapted (e.g., tagmented) nucleic acid fragment from the biologicalsample to the array; and (e) capturing the adapted (e.g., tagmented)nucleic acid fragment with the capture domain.

Preparation of the Biological Sample

Biological Samples and Analytes

The biological sample as used herein can be any suitable biologicalsample described herein or known in the art. In some embodiments, thebiological sample is a tissue. In some embodiments, the biologicalsample is a tissue section. In some embodiments, the tissue isflash-frozen and sectioned. Any suitable methods described herein orknown in the art can be used to flash-freeze and section the tissuesample. In some embodiments, the biological sample, e.g., the tissue, isflash-frozen using liquid nitrogen before sectioning. In someembodiments, the sectioning is performed using cryosectioning. In someembodiments, the methods further comprises a thawing step, after thecryosectioning. In some embodiments, the biological sample, e.g., thetissue sample is fixed, for example in methanol, acetone, PFA or isformalin-fixed and paraffin-embedded (FFPE).

The biological sample, e.g., tissue sample, can be stained, and imagedprior, during, and/or after each step of the methods described herein.Any of the methods described herein or known in the art can be used tostain and/or image the biological sample. In some embodiments, theimaging occurs prior to deaminating the sample. In some embodiments, thebiological sample is stained using an H&E staining method. In someembodiments, the tissue sample is stained and imaged for about 10minutes to about 2 hours (or any of the subranges of this rangedescribed herein). Additional time may be needed for staining andimaging of different types of biological samples.

The tissue sample can be obtained from any suitable location in a tissueor organ of a subject, e.g., a human subject. In some embodiments, thetissue sample is obtained from a location where the DNA isdifferentially methylated compared to a reference location. The locationwhere the DNA is differentially methylated can be, for example, adiseased cell, tissue or organ, an infected cell, tissue or organ, adamaged cell, tissue or organ, a cancerous cell, tissue or organ (e.g.,a tumor cell, tissue, or organ), or a differentiating cell, tissue, ororgan (e.g., a stem cell or a tissue or organ that comprises one or morestem cells). In some embodiments, the location wherein the DNA isdifferentially methylated is a cell, tissue, or organ that has beenadministered one or more drug(s), e.g., therapeutic drugs. Otherlocations that include differentially methylated DNA are known in theart.

In some embodiments, the biological sample is a tumor cell, tissue, ororgan. Non-limiting examples of cancers referred to in any one themethods described herein include: sarcomas, carcinomas, adrenocorticalcarcinoma, AIDS-related cancers, anal cancer, appendix cancer,astrocytomas, atypical teratoid/rhabdoid tumor, basal cell carcinoma,bladder cancer, brain stem glioma, brain tumors (including brain stemglioma, central nervous system atypical teratoid/rhabdoid tumor, centralnervous system embryonal tumors, astrocytomas, craniopharyngioma,ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma,pineal parenchymal tumors of intermediate differentiation,supratentorial primitive neuroectodermal tumors, and pineoblastoma),breast cancer, bronchial tumors, cancer of unknown primary site,carcinoid tumor, carcinoma of unknown primary site, central nervoussystem atypical teratoid/rhabdoid tumor, central nervous systemembryonal tumors, cervical cancer, childhood cancers, chordoma, coloncancer, colorectal cancer, craniopharyngioma, endocrine pancreas isletcell tumors, endometrial cancer, ependymoblastoma, ependymoma,esophageal cancer, esthesioneuroblastoma, Ewing sarcoma, extracranialgerm cell tumor, extragonadal germ cell tumor, extrahepatic bile ductcancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinalcarcinoid tumor, gastrointestinal stromal cell tumor, gastrointestinalstromal tumor (GIST), gestational trophoblastic tumor, glioma, head andneck cancer, heart cancer, hypopharyngeal cancer, intraocular melanoma,islet cell tumors, Kaposi's sarcoma, kidney cancer, Langerhans cellhistiocytosis, laryngeal cancer, lip cancer, liver cancer, lung cancer,malignant fibrous histiocytoma bone cancer, medulloblastoma,medulloepithelioma, melanoma, Merkel cell carcinoma, Merkel cell skincarcinoma, mesothelioma, metastatic squamous neck cancer with occultprimary, mouth cancer, multiple endocrine neoplasia syndromes, multiplemyeloma, multiple myeloma/plasma cell neoplasm, mycosis fungoides,myelodysplastic syndromes, myeloproliferative neoplasms, nasal cavitycancer, nasopharyngeal cancer, neuroblastoma, non-melanoma skin cancer,non-small cell lung cancer, oral cancer, oral cavity cancer,oropharyngeal cancer, osteosarcoma, other brain and spinal cord tumors,ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor,ovarian low malignant potential tumor, pancreatic cancer,papillomatosis, paranasal sinus cancer, parathyroid cancer, pelviccancer, penile cancer, pharyngeal cancer, pineal parenchymal tumors ofintermediate differentiation, pineoblastoma, pituitary tumor,pleuropulmonary blastoma, primary hepatocellular liver cancer, prostatecancer, rectal cancer, renal cancer, renal cell (kidney) cancer, renalcell cancer, respiratory tract cancer, retinoblastoma, rhabdomyosarcoma,salivary gland cancer, Sezary syndrome, small cell lung cancer, smallintestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamousneck cancer, stomach (gastric) cancer, supratentorial primitiveneuroectodermal tumors, testicular cancer, throat cancer, thymiccarcinoma, thymoma, thyroid cancer, transitional cell cancer,transitional cell cancer of the renal pelvis and ureter, trophoblastictumor, ureter cancer, urethral cancer, uterine cancer, uterine sarcoma,vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilm'stumor.

In some embodiments, the biological sample is a cancer sample. In someembodiments, the cancer can be treated using an epigenetic therapy(e.g., a hypomethylating agent).

In some embodiments, the cancer is a non-small-cell lung cancer (NSCLC),a microsatellite-stable colorectal cancer (CRC), a head and neck cancer,a head and neck squamous cell carcinoma (HNSCC), a melanoma, an acutemyeloid leukemia (AML), a myelodysplastic syndromes (MDS), a pancreaticductal adenocarcinoma (PDAC), an ovarian cancer, a primary peritoneal orfallopian tube cancer, a peripheral T-Cell lymphoma (PTCL), an ovariancancer type II or oestrogen receptor-positive and HER2-negative breastcancer, a diffuse large B-cell lymphoma (DLBCL), a central nervoussystem (CNS) solid tumor, a lung cancer, a renal cancer, ahepatocellular carcinoma, a pancreatic adenocarcinoma, acholangiocarcinoma, or a chronic myelomonocytic leukemia (CMML).

Methylation of key markers are used for cancer diagnosis. These keymarkers are described, e.g., in Locke W J et al., DNA Methylation CancerBiomarkers: Translation to the Clinic. Front. Genet. 10:1150, 2019;Nassiri, F. et al., Detection and discrimination of intracranial tumorsusing plasma cell-free DNA methylomes. Nat Med 26, 1044-1047, 2020; andNuzzo, P. V. et al., Detection of renal cell carcinoma using plasma andurine cell-free DNA methylomes. Nat Med 26, 1041-1043 (2020), the entirecontents of which are incorporated herein by reference.

Suitable agents such as hypomethylating agents can be used as epigenetictherapies for the treatment of cancer. 5-Azacitidine (5-Aza),5-aza-2′-deoxycytidine (decitabine) and SGI-110 (guadecitabine) areanalogues of the nucleoside cytidine that irreversibly sequester DNMTproteins to DNA, leading to global DNA hypomethylation.

In some embodiments, the cancer sample is from a subject treated with acancer therapy. In some embodiments, the cancer therapy is therapy withnucleoside cytidine analogue. In some embodiments, the cancer therapy isa therapy with 5-Azacitidine (5-Aza), 5-aza-2′-deoxycytidine(decitabine), or SGI-110 (guadecitabine).

The analyte can be any suitable analyte described herein. In someembodiments, the analyte is a nucleic acid. In some embodiments, theanalyte is a DNA. In some embodiments, the analyte is a genomic DNA. Insome embodiments, the analyte is a non-genomic DNA. In some embodiments,the DNA is within a coding region of a gene. In some embodiments, theDNA is within a promoter region of a gene. In some embodiments, the DNAis outside of the coding region of a gene. In some embodiments, the DNAspans a coding region and a non-coding region of a gene. In someembodiments, the DNA spans the coding region and/or the non-codingregion of more than one genes.

In some embodiments, the DNA is a methylated DNA, e.g., a DNA comprisingone or more methylated cytosines. In some embodiments, the DNA is anunmethylated DNA, e.g., a DNA that does not comprise any methylatedcytosine. In some embodiments, the DNA is a DNA that is differentiallymethylated in a location compared to a reference location.

The size of the analyte, e.g., DNA, can be any suitable size of anucleic acid molecule in a biological sample. In some embodiments, thesize of the target nucleic acid is about 50 nucleotides to about 100,000nucleotides (e.g., about 50 nucleotides to about 200 nucleotides, about200 nucleotides to about 500 nucleotides, about 500 nucleotides to about1,000 nucleotides, about 1,000 nucleotides to about 2,000 nucleotides,about 2,000 nucleotides to about 4,000 nucleotides, about 4,000nucleotides to about 6,000 nucleotides, about 6,000 nucleotides to about8,000 nucleotides, about 8,000 nucleotides to about 10,000 nucleotides,about 10,000 nucleotides to about 20,000 nucleotides, about 20,000nucleotides to about 30,000 nucleotides, about 30,000 nucleotides toabout 40,000 nucleotides, about 40,000 nucleotides to about 50,000nucleotides, about 50,000 nucleotides to about 60,000 nucleotides, about60,000 nucleotides to about 70,000 nucleotides, about 70,000 nucleotidesto about 80,000 nucleotides, about 80,000 nucleotides to about 90,000nucleotides, or about 90,000 nucleotides to about 100,000 nucleotides).

In some embodiments, the DNA includes at least a portion of a tumorbiomarker gene. In some embodiments, the tumor biomarker is a tumorantigen. Exemplary tumor antigens include, but are not limited to,melanoma-associated antigen (MAGE) series of antigens (e.g., MAGE-Ci(cancer/testis antigen CT7), MAGE-B1 antigen (MAGE-XP antigen, DAM10),MAGE-B2 antigen (DAM6), MAGE-2 antigen, MAGE-4a antigen, and MAGE-4bantigen), tyrosinase, glycoprotein 100 (gp100), disialoganglioside GD-2,disialoganglioside O-acetylated GD-3, ganglioside GM-2, epidermal growthfactor receptor (EGFR), vascular endothelial growth factor receptor(VEGFR), mutant B-Raf antigen associated with melanoma and colon cancer,human epidermal growth factor receptor-2 (HER-2/neu) antigen,melanoma-associated antigen recognized by T cells (MART-1) (e.g., MART-126-35 peptide or MART-1 27-35 peptide), protein kinase C-bindingprotein, reverse transcriptase protein, A-kinase-anchoring protein (AKAPprotein), vaccinia-related kinase Serine/Threonine Kinase 1 (VRK1),fucosyltransferase (T6-7), zinc finger protein 258 (T11-6), p53-bindingprotein (T1-52), T5-15 (KIAA1735), T5-13 (Sos1), T11-5 (hypotheticalprotein MGC4170), T11-9 (hypothetical protein AF225417), T11-3 (trapankyrin repeat), T7-1 (KIAA1288), a mutant or wild type RAS peptide,Homo sapiens telomerase ferment (hTRT), cytokeratin-19 (CYFRA21-1),squamous cell carcinoma antigen 1 (SCCA-1), protein T4-A, squamous cellcarcinoma antigen 2 (SCCA-2), ovarian carcinoma antigen CA125 (1A1-3B)(KIAA0049), cell surface-associated MUCIN 1 (e.g., tumor-associatedMUCIN, carcinoma-associated MUCIN, polymorphic epithelial MUCINpeanut-reactive urinary MUCIN, polymorphic epithelial mucin (PEM), PEMT,episialin, tumor-associated epithelial membrane antigen, epithelialmembrane antigen (EMA), H23 antigen (H23AG), PUM, and breastcarcinoma-associated antigen DF3), CTCL tumor antigen se1-1, CTCL tumorantigen se14-3, CTCL tumor antigen se20-4, CTCL tumor antigen se20-9,CTCL tumor antigen se33-1, CTCL tumor antigen se37-2, CTCL tumor antigense57-1, CTCL tumor antigen se89-1, prostate-specific membrane antigen,5T4 oncofetal trophoblast glycoprotein, Orf73 Kaposi'ssarcoma-associated herpesvirus, colon cancer antigen NY-CO-45, lungcancer antigen NY-LU-12 variant A, cancer associated surface antigen,adenocarcinoma antigen ART1, paraneoplastic associatedbrain-testis-cancer antigen (onconeuronal antigen MA2; paraneoplasticneuronal antigen), neuro-oncological ventral antigen 2 (NOVA2),hepatocellular carcinoma antigen gene 520, tumor-associated antigenCO-029, tumor-associated antigen MAGE-X2, synovial sarcoma antigen, Xbreakpoint 2, squamous cell carcinoma antigen recognized by T cell,serologically defined colon cancer antigen 1, serologically definedbreast cancer antigen NY-BR-15, serologically defined breast cancerantigen NY-BR-16, chromogranin A, parathyroid secretory protein 1,pancreatic cancer-associated antigen (DUPAN-2), carbohydrate antigen CA19-9, carbohydrate antigen CA 72-4, carbohydrate antigen CA 195, andcarcinoembryonic antigen (CEA).

In some embodiments, the tumor antigen is BRCA1, CDKN2A (p16^(INK4a)),CDKN2B (p15^(INK4b)) GSTP1, MGMT, RASSF1A, or SFRP2.

Exemplary First and Second Substrates

In some instances, the biological sample is placed (e.g., mounted orotherwise immobilized) on a first substrate. The first substrate can beany solid or semi-solid support upon which a biological sample can bemounted. In some instances, the first substrate is a slide. In someinstances, the slide is a glass slide. In some embodiments, thesubstrate is made of glass, silicon, paper, hydrogel, polymer monoliths,or other material known in the art. In some embodiments, the firstsubstrate is comprised of an inert material or matrix (e.g., glassslides) that has been functionalized by, for example, treating thesubstrate with a material comprising reactive groups which facilitatemounting of the biological sample.

In some embodiments, the first substrate does not comprise a plurality(e.g., array) of capture probes, each comprising a spatial barcode.

A substrate, e.g., a first substrate and/or a second substrate, cangenerally have any suitable form or format. For example, a substrate canbe flat, curved, e.g., convexly or concavely curved. For example, afirst substrate can be curved towards the area where the interactionbetween a biological sample, e.g., tissue sample, and a first substratetakes place. In some embodiments, a substrate is flat, e.g., planar,chip, or slide. A substrate can contain one or more patterned surfaceswithin the first substrate (e.g., channels, wells, projections, ridges,divots, etc.).

A substrate, e.g., a first substrate and/or second substrate, can be ofany desired shape. For example, a substrate can be typically a thin,flat shape (e.g., a square or a rectangle). In some embodiments, asubstrate structure has rounded corners (e.g., for increased safety orrobustness). In some embodiments, a substrate structure has one or morecut-off corners (e.g., for use with a slide clamp or cross-table). Insome embodiments wherein a substrate structure is flat, the substratestructure can be any appropriate type of support having a flat surface(e.g., a chip or a slide such as a microscope slide).

First and/or second substrates can optionally include various structuressuch as, but not limited to, projections, ridges, and channels. Asubstrate can be micropatterned to limit lateral diffusion of analytes(e.g., to improve resolution of the spatial analysis). A substratemodified with such structures can be modified to allow association ofanalytes, features (e.g., beads), or probes at individual sites. Forexample, the sites where a substrate is modified with various structurescan be contiguous or non-contiguous with other sites.

In some embodiments, the surface of a first and/or second substrate ismodified to contain one or more wells, using techniques such as (but notlimited to) stamping, microetching, or molding techniques. In someembodiments in which a first and/or second substrate includes one ormore wells, the first substrate can be a concavity slide or cavityslide. For example, wells can be formed by one or more shallowdepressions on the surface of the first and/or second substrate. In someembodiments, where a first and/or second substrate includes one or morewells, the wells can be formed by attaching a cassette (e.g., a cassettecontaining one or more chambers) to a surface of the first substratestructure.

In some embodiments where the first and/or second substrate is modifiedto contain one or more structures, including but not limited to, wells,projections, ridges, features, or markings, the structures can includephysically altered sites. For example, a first and/or second substratemodified with various structures can include physical properties,including, but not limited to, physical configurations, magnetic orcompressive forces, chemically functionalized sites, chemically alteredsites, and/or electrostatically altered sites. In some embodiments wherethe first substrate is modified to contain various structures, includingbut not limited to wells, projections, ridges, features, or markings,the structures are applied in a pattern. Alternatively, the structurescan be randomly distributed.

In some embodiments, a first substrate includes one or more markings onits surface, e.g., to provide guidance for aligning at least a portionof the biological sample with a plurality of capture probes on thesecond substrate during a sandwich type process disclosed herein. Forexample, the first substrate can include a sample area indicatoridentifying the sample area. In some embodiments, the sample areaindicator on the first substrate is aligned with an area of the secondsubstrate comprising a plurality of capture probes. In some embodiments,the first and/or second substrate can include a fiducial mark. In someembodiments, the first and/or second substrate does not comprise afiducial mark. In some embodiments, the first substrate does notcomprise a fiducial mark and the second substrate comprises a fiducialmark. Such markings can be made using techniques including, but notlimited to, printing, sand-blasting, and depositing on the surface.

In some embodiments, imaging can be performed using one or more fiducialmarkers, i.e., objects placed in the field of view of an imaging systemwhich appear in the image produced. Fiducial markers are typically usedas a point of reference or measurement scale. Fiducial markers caninclude, but are not limited to, detectable labels such as fluorescent,radioactive, chemiluminescent, and colorimetric labels. The use offiducial markers to stabilize and orient biological samples isdescribed, for example, in Carter et al., Applied Optics 46:421-427,2007), the entire contents of which are incorporated herein byreference. In some embodiments, a fiducial marker can be a physicalparticle (e.g., a nanoparticle, a microsphere, a nanosphere, a bead, apost, or any of the other exemplary physical particles described hereinor known in the art).

In some embodiments, a fiducial marker can be present on a firstsubstrate to provide orientation of the biological sample. In someembodiments, a microsphere can be coupled to a first substrate to aid inorientation of the biological sample. In some examples, a microspherecoupled to a first substrate can produce an optical signal (e.g.,fluorescence). In some embodiments, a quantum dot can be coupled to thefirst substrate to aid in the orientation of the biological sample. Insome examples, a quantum dot coupled to a first substrate can produce anoptical signal.

In some embodiments, a fiducial marker can be an immobilized moleculewith which a detectable signal molecule can interact to generate asignal. For example, a marker nucleic acid can be linked or coupled to achemical moiety capable of fluorescing when subjected to light of aspecific wavelength (or range of wavelengths). Although not required, itcan be advantageous to use a marker that can be detected using the sameconditions (e.g., imaging conditions) used to detect a labelled cDNA.

In some embodiments, a fiducial marker can be randomly placed in thefield of view. For example, an oligonucleotide containing a fluorophorecan be randomly printed, stamped, synthesized, or attached to a firstsubstrate (e.g., a glass slide) at a random position on the firstsubstrate. A tissue section can be contacted with the first substratesuch that the oligonucleotide containing the fluorophore contacts, or isin proximity to, a cell from the tissue section or a component of thecell (e.g., an mRNA or DNA molecule). An image of the first substrateand the tissue section can be obtained, and the position of thefluorophore within the tissue section image can be determined (e.g., byreviewing an optical image of the tissue section overlaid with thefluorophore detection). In some embodiments, fiducial markers can beprecisely placed in the field of view (e.g., at known locations on afirst substrate). In this instance, a fiducial marker can be stamped,attached, or synthesized on the first substrate and contacted with abiological sample. Typically, an image of the sample and the fiducialmarker is taken, and the position of the fiducial marker on the firstsubstrate can be confirmed by viewing the image.

In some embodiments, a fiducial marker can be an immobilized molecule(e.g., a physical particle) attached to the first substrate. Forexample, a fiducial marker can be a nanoparticle, e.g., a nanorod, ananowire, a nanocube, a nanopyramid, or a spherical nanoparticle. Insome examples, the nanoparticle can be made of a heavy metal (e.g.,gold).

A wide variety of different first substrates can be used for theforegoing purposes. In general, a first substrate can be any suitablesupport material. Exemplary first substrates include, but are notlimited to, glass, modified and/or functionalized glass, hydrogels,films, membranes, plastics (including e.g., acrylics, polystyrene,copolymers of styrene and other materials, polypropylene, polyethylene,polybutylene, polyurethanes, Teflon™, cyclic olefins, polyimides etc.),nylon, ceramics, resins, Zeonor, silica or silica-based materialsincluding silicon and modified silicon, carbon, metals, inorganicglasses, optical fiber bundles, and polymers, such as polystyrene,cyclic olefin copolymers (COCs), cyclic olefin polymers (COPs),polypropylene, polyethylene polycarbonate, or combinations thereof.

Among the examples of first substrate materials discussed above,polystyrene is a hydrophobic material suitable for binding negativelycharged macromolecules because it normally contains few hydrophilicgroups. For nucleic acids immobilized on glass slides, by increasing thehydrophobicity of the glass surface the nucleic acid immobilization canbe increased. Such an enhancement can permit a relatively more denselypacked formation (e.g., provide improved specificity and resolution).

In another example, a first substrate can be a flow cell. Flow cells canbe formed of any of the foregoing materials, and can include channelsthat permit reagents, solvents, features, and analytes to pass throughthe flow cell. In some embodiments, a hydrogel embedded biologicalsample is assembled in a flow cell (e.g., the flow cell is utilized tointroduce the hydrogel to the biological sample). In some embodiments, ahydrogel embedded biological sample is not assembled in a flow cell. Insome embodiments, the hydrogel embedded biological sample can then beprepared and/or isometrically expanded as described herein.

Exemplary substrates similar to the first substrate (e.g., a substratehaving no capture probes) and/or the second substrate are described inWO 2020/123320, which is hereby incorporated by reference in itsentirety.

Staining and Imaging the Biological Sample

After placement of the biological sample onto the first substrate,biological samples can be stained using a wide variety of stains andstaining techniques. In some embodiments, a sample can be stained usingany number of biological stains, including but not limited to, acridineorange, Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI,eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains,iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red,osmium tetroxide, propidium iodide, rhodamine, or safranin. In someinstances, the methods disclosed herein include imaging the biologicalsample. In some instances, imaging the sample occurs prior todeaminating the biological sample.

The sample can be stained using known staining techniques, includingCan-Grunwald, Giemsa, hematoxylin and eosin (H&E), Jenner's, Leishman,Masson's trichrome, Papanicolaou, Romanowsky, silver, Sudan, Wright's,and/or Periodic Acid Schiff (PAS) staining techniques. PAS staining istypically performed after formalin or acetone fixation. In someinstances, the stain is an H&E stain.

In some embodiments, the biological sample can be stained using adetectable label (e.g., radioisotopes, fluorophores, chemiluminescentcompounds, bioluminescent compounds, and dyes) as described elsewhereherein. In some embodiments, a biological sample is stained using onlyone type of stain or one technique. In some embodiments, stainingincludes biological staining techniques such as H&E staining. In someembodiments, staining includes identifying analytes usingfluorescently-conjugated antibodies. In some embodiments, a biologicalsample is stained using two or more different types of stains, or two ormore different staining techniques. For example, a biological sample canbe prepared by staining and imaging using one technique (e.g., H&Estaining and brightfield imaging), followed by staining and imagingusing another technique (e.g., IHC/IF staining and fluorescencemicroscopy) for the same biological sample.

In some embodiments, biological samples can be destained. Methods ofdestaining or discoloring a biological sample are known in the art, andgenerally depend on the nature of the stain(s) applied to the sample.For example, H&E staining can be destained by washing the sample in HCl,or any other acid (e.g., selenic acid, sulfuric acid, hydroiodic acid,benzoic acid, carbonic acid, malic acid, phosphoric acid, oxalic acid,succinic acid, salicylic acid, tartaric acid, sulfurous acid,trichloroacetic acid, hydrobromic acid, hydrochloric acid, nitric acid,orthophosphoric acid, arsenic acid, selenous acid, chromic acid, citricacid, hydrofluoric acid, nitrous acid, isocyanic acid, formic acid,hydrogen selenide, molybdic acid, lactic acid, acetic acid, carbonicacid, hydrogen sulfide, or combinations thereof). In some embodiments,destaining can include 1, 2, 3, 4, 5, or more washes in an acid (e.g.,HCl). In some embodiments, destaining can include adding HCl to adownstream solution (e.g., permeabilization solution). In someembodiments, destaining can include dissolving an enzyme used in thedisclosed methods (e.g., pepsin) in an acid (e.g., HCl) solution. Insome embodiments, after destaining hematoxylin with an acid, otherreagents can be added to the destaining solution to raise the pH for usein other applications. For example, SDS can be added to an aciddestaining solution in order to raise the pH as compared to the aciddestaining solution alone. As another example, in some embodiments, oneor more immunofluorescence stains are applied to the sample via antibodycoupling. Such stains can be removed using techniques such as cleavageof disulfide linkages via treatment with a reducing agent and detergentwashing, chaotropic salt treatment, treatment with antigen retrievalsolution, and treatment with an acidic glycine buffer. Methods formultiplexed staining and destaining are described, for example, inBolognesi et al., J. Histochem. Cytochem. 2017; 65(8): 431-444, Lin etal., Nat Commun. 2015; 6:8390, Pirici et al., J. Histochem. Cytochem.2009; 57:567-75, and Glass et al., J. Histochem. Cytochem. 2009;57:899-905, the entire contents of each of which are incorporated hereinby reference.

In some embodiments, immunofluorescence or immunohistochemistryprotocols (direct and indirect staining techniques) can be performed asa part of, or in addition to, the exemplary spatial workflows presentedherein. For example, tissue sections can be fixed according to methodsdescribed herein. The biological sample can be transferred to an array(e.g., capture probe array), wherein analytes (e.g., proteins) areprobed using immunofluorescence protocols. For example, the sample canbe rehydrated, blocked, and permeabilized (3×SSC, 2% BSA, 0.1% Triton X,1 U/μl RNAse inhibitor for 10 min at 4° C.) before being stained withfluorescent primary antibodies (1:100 in 3×SSC, 2% BSA, 0.1% Triton X, 1U/μl RNAse inhibitor for 30 min at 4° C.). The biological sample can bewashed, coverslipped (in glycerol+1 U/μl RNAse inhibitor), imaged (e.g.,using a confocal microscope or other apparatus capable of fluorescentdetection), washed, and processed according to analyte capture orspatial workflows described herein.

As used herein, an antigen retrieval buffer can improve antibody capturein IF/IHC protocols. An exemplary protocol for antigen retrieval can bepreheating the antigen retrieval buffer (e.g., to 95° C.), immersing thebiological sample in the heated antigen retrieval buffer for apredetermined time, and then removing the biological sample from theantigen retrieval buffer and washing the biological sample.

In some embodiments, optimizing permeabilization can be useful foridentifying intracellular analytes. Permeabilization optimization caninclude selection of permeabilization agents, concentration ofpermeabilization agents, and permeabilization duration. Tissuepermeabilization is discussed elsewhere herein.

In some embodiments, blocking an array and/or a biological sample inpreparation of labeling the biological sample decreases unspecificbinding of the antibodies to the array and/or biological sample(decreases background). Some embodiments provide for blockingbuffers/blocking solutions that can be applied before and/or duringapplication of the label, wherein the blocking buffer can include ablocking agent, and optionally a surfactant and/or a salt solution. Insome embodiments, a blocking agent can be bovine serum albumin (BSA),serum, gelatin (e.g., fish gelatin), milk (e.g., non-fat dry milk),casein, polyethylene glycol (PEG), polyvinyl alcohol (PVA), orpolyvinylpyrrolidone (PVP), biotin blocking reagent, a peroxidaseblocking reagent, levamisole, Carnoy's solution, glycine, lysine, sodiumborohydride, pontamine sky blue, Sudan Black, trypan blue, FITC blockingagent, and/or acetic acid. The blocking buffer/blocking solution can beapplied to the array and/or biological sample prior to and/or duringlabeling (e.g., application of fluorophore-conjugated antibodies) to thebiological sample.

Deamination of Nucleic Acids in the Biological Sample

In some embodiments, the methods described herein include deaminatingthe analyte. In some embodiments, either before or after deamination,the methods described herein include a step of permeabilizing thebiological sample. In some instances, the permeabilization step occursprior to deaminating the analyte in the sample. In some instances,permeabilizing includes contacting the biological sample with apermeabilization reagent. Any suitable permeabilization reagentdescribed herein can be used. In some embodiments, the permeabilizationreagent is an endopeptidase. Endopeptidases that can be used include butare not limited to trypsin, chymotrypsin, elastase, thermolysin, pepsin,clostripan, glutamyl endopeptidase (GluC), ArgC, peptidyl-aspendopeptidase (ApsN), endopeptidase LysC and endopeptidase LysN. In someembodiments, the endopeptidase is pepsin.

In some embodiments, the deaminating of the analyte is achieved bycontacting the analyte with one or more enzymatic or chemicaldeaminating agent(s). In some instances, the deaminating agent fragmentsthe analyte (e.g., DNA). Any suitable deaminating agents can be used inthe methods described herein. In some embodiments, the analyte, e.g.,DNA, is denatured into a single-stranded molecule prior to the treatmentof the deaminating agent(s). In some embodiments, the deaminating stepincludes contacting the sample with a composition comprising bisulfite.In some embodiments, the bisulfite is sodium bisulfite.

In some instances, the deaminating step is performed prior to thehybridization of one or more probes to the analytes (e.g., nucleic acidmolecules) and the capture of the ligated (e.g., ligation) probes on aspatial array. For example, the biological sample is placed on a regularslide (different from a spatial array) without capture probes, and thenucleic acid molecules in the biological sample are deaminated asdescribed herein.

In some embodiments, the deaminating comprises treating the sampleenzymatically. In some embodiments, the sample is treated with adeaminase. Any suitable enzymes known in the art can be used in themethods described herein. In some embodiments, the enzyme is aeukaryotic DNA methyltransferases (CG). In some embodiments, the enzymeis a prokaryotic DNA(cytosine-5)methyltransferase. In some embodiments,the enzyme is apolipoprotein B mRNA editing enzyme, catalyticpolypeptide-like (APOBEC, cytidine deaminase). In some embodiments, theenzyme is an enzyme of the ten-eleven translocation (TET) familydemethylase.

In some embodiments, unmethylated cytosines, if present in the analytein the sample, are deaminated into uracils. After the binding of theprobe with the deaminated analyte, the probe is extended wherein eachuracil, if present in the analyte, is base-paired with an adenine. Insome embodiments, the extended probe comprises an adenine in eachposition corresponding to the unmethylated cytosines in the analyte inthe sample. In some embodiments, all of the unmethylated cytosines inthe analyte are converted to uracils by the deaminating step. In someinstances, cytosines that are methylated are not converted to uracil.Instead, methylated cytosines remain as cytosines.

In some instances, prior to addition of the probes described in the nextsection, the sample is treated with a permeabilization agent. In someinstances, the permeabilization agent is a diluted permeabilizationagent. In some instances, the diluted permeabilization agent includes,but is not limited to, organic solvents (e.g., acetone, ethanol, andmethanol), cross-linking agents (e.g., paraformaldehyde), detergents(e.g., saponin, Triton X-100™, Tween-20™, or sodium dodecyl sulfate(SDS)), and enzymes (e.g., trypsin, proteases (e.g., proteinase K). Insome embodiments, the detergent is an anionic detergent (e.g., SDS orN-lauroylsarcosine sodium salt solution). Exemplary permeabilizationreagents are described in in PCT Patent Application Publication No. WO2020/123320, which is incorporated by reference in its entirety. In someinstances, dilution of the permeabilization agent is by about 10-fold,about 50-fold, about 100-fold, about 200-fold, about 300-fold, about400-fold, about 500-fold, or higher.

In some embodiments, the method further comprises washing the biologicalsample. In some instances, a wash step occurs between the deaminationstep and the step where the probes are added. In some embodiments, thewashing step is conducted to remove all or a part of the deaminatingagent. In some embodiments, the washing step does not remove the analytein the biological sample. In some embodiments, the washing step removesan insignificant amount of the analyte in the biological sample. In someinstances, a wash step occurs after hybridizing the oligonucleotides. Insome instances, this wash step removes any unbound oligonucleotides andcan be performed using any technique or solution disclosed herein orknown in the art. In some embodiments, multiple wash steps are performedto remove unbound oligonucleotides.

Addition of Probes to Biological Sample

Addition of First and Second Probes

After deamination, in some instances, one or more probes are added tothe biological sample. In some instances, the one or more probes (e.g.,a first probe; a second probe) is at least 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99. 100, at least about 105, at leastabout 110, at least about 115, at least about 120, at least about 125,at least about 135, at least about 140, or more nucleotides in length.In some embodiments, the probe includes sequences that are complementaryor substantially complementary to an analyte, e.g., a nucleic acid. Bysubstantially complementary, it is meant that the probe is at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% complementary to asequence in an analyte. In some instances, the first probe and thesecond probe hybridize to adjacent sequences on an analyte.

In some instances, the probes hybridize to a sequence that is from 5 to140 nucleotides in length (e.g., is about 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99. 100, 101, 102,103, 104, 105, 106, 107, 108. 109. 110, 111, 112, 113, 114, 115, 116,117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,131, 132, 133, 134, 135, 136, 137, 138, 139, or 140 nucleotides inlength). In some instances, the probes hybridize to a sequence that isfrom 15 to 120 nucleotides in length.

In some instances, the probes are DNA probes. In some instances, a firstprobe includes at least two ribonucleic acid bases at the 3′ end and asecond probe includes a phosphorylated nucleotide at the 5′ end.

Probes (e.g., a first probe and a second probe) can be designed usingmethods known in the art. In addition, probes can be designed so that(1) one or both probes hybridize directly to a potential region ofmethylation or (2) the probes flank a potential region of methylation.

In some instances, the probes used herein are designed to hybridize tosequences that are mutated (e.g., from cytosine to thymine) as a resultof the deamination step (i.e., deaminated nucleic acid). In someinstances, the probes used herein are designed to hybridize to sequencesthat are not mutated (e.g., cytosines that are methylated are notmutated during the deamination step) as a result of the deaminationstep. In some instances, the probes used herein are designed tohybridize to sequences that include at least one nucleotide that ismutated as a result of the deamination step. In some instances, theprobes used herein are designed to hybridize to sequences that includemore than one (e.g., 2, 3, 4, 5, or more) nucleotide that is mutated asa result of the deamination step.

In some instances, probes are designed to flank (i.e., surround)sequences that are sites of methylation investigation. In someinstances, the sequence between the two regions of an analyte where theprobes hybridize (i.e., the intervening sequence) include one or morenucleotides that is mutated (e.g., from cytosine to thymine) during thedeamination step. In some instances, the intervening sequence includesat least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides that have beenmutated during the deamination step described herein. For example, insome instances, a first probe can be designed to hybridize to a site 5′to a site of methylation investigation, and a second probe can bedesigned to hybridize to a site 5′ to a site of methylationinvestigation (or vice versa). One hybridized, the sequence in betweenthe two sites of hybridization can be determined, providing insight onwhether a particular nucleotide (e.g., a cytosine) has been methylated.In some instances, the probes hybridize to sequences that are at leastabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides apart. In someembodiments, at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)deaminated nucleotide is located in the sequence between the hybridizedprobes.

In some instances, more than one (e.g., 2, 3, 4, 5, or more) probeshybridize to a target DNA molecule. In some instances, two probeshybridize to sequences that are adjacent to one another. In someinstances, the probes are coupled. In some instances, the probes arecoupled via ligation. In some instances, because the probes hybridize toadjacent sequences, ligation can occur between the adjacent probes afterhybridization (i.e., no extension step as described herein is required).In some instances, two probes hybridize to sequences of an analyte thatare separated by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, about 15, about20, about 25, about 30, about 35, about 40, about 45, about 50, about55, about 60, about 65, about 70, about 75, about 80, about 85, about90, about 95, about 100, about 125, about 150, about 175, about 200,about 250, about 300, about 350, about 400, about 450, about 500, about600, about 700, about 800, about 900, or about 1000 nucleotides. In someinstances, the probes hybridize to a sequence that has been modified(e.g. includes a mutation) after deamination. In some instances, themutation is a cytosine to a thymine. In some instances, the analyteincludes more than one mutation as a result of the deamination step. Insome instances, there are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or morecytosines that are mutated to a thymine (i.e., the cytosines are notmethylated). In some instances, there are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more cytosines that are not mutated to a thymine (i.e., the cytosinesare methylated). In some instances, the probe is designed to detect oneor more deamination mutations.

The one or more probes include a sequence that specifically binds to asequence on the target analyte. In some embodiments, the binding moietycomprises a sequence that specifically binds to a region that does nothave a CG dinucleotide. In some instances, the binding moiety comprisesa sequence that specifically binds to a region that includes AA, AT, AC,AG, CA, CT, CC, GA, GC, GT, GG, TA, TC, TG, or TT dinucleotides. In someembodiments, the binding of the binding moiety to the target analytedoes not affect the identification of the methylation status of thetarget analyte.

In some instances, the one or more probes (e.g., a first probe; a secondprobe) further includes a sequence that can hybridize to a capture probesequence (e.g., on an array, as described herein). In some instances,the sequence that hybridizes to the capture probe is a poly-adenylation(poly(A)) sequence. In some instances, the sequence that hybridizes tothe capture probe is a degenerate (e.g., random) sequence. In someinstances, the sequence that hybridizes to the capture probe is designedto be a specific sequence, for example a sequence that is complementaryto a specific target sequence in a DNA or RNA molecule.

In some embodiments, at least one of the probes is extended using apolymerase. In some instances, the methods disclosed herein includegenerating an extended first probe. In some instances, the methodsdisclosed herein include generating an extended second probe. In someinstances, the extended first probe or the extended second probeincludes a sequence complementary to a sequence between the firstsequence and the second sequence. In some instances, one of the probesis extended to fill in the gap between the two hybridized probes. Insome embodiments, the capture probe is extended using a polymerase. Insome embodiments, the polymerase is a DNA polymerase. In someembodiments, the DNA polymerase is a thermostable DNA polymeraseincluding, but are not limited to, DNA polymerase such as Phusion, HotStart Taq DNA Polymerase, and EpiMark® Hot Start Taq DNA Polymerase.

In some instances, the probes (e.g., the first probe and the secondprobe) are ligated together. In instances of ligation, any suitableligase can be used in the methods described herein. In some instances,the probes may be subjected to an enzymatic ligation reaction, using aligase (e.g., T4 RNA ligase 2, a splintR ligase, a single stranded DNAligase). In some instances, the ligase is T4 DNA ligase. In someinstances, the ligase is T4 RNA ligase 2, also known as Rnl2, whichligates the 3′ hydroxyl end of a RNA to the 5′ phosphate of DNA in adouble stranded structure. T4 DNA ligase is an enzyme belonging to theDNA ligase family of enzymes that catalyzes the formation of a covalentphosphodiester bond from a free 3′ hydroxyl group on one DNA moleculeand a free 5′ phosphate group of a second, separate DNA molecule, thuscovalently linking the two DNA strands together to form a single DNAstrand. In some instances, the ligase is splintR ligase. SplintR Ligase,also known as PBCV-1 DNA Ligase or Chorella virus DNA Ligase,efficiently catalyzes the coupling (e.g., ligation) of adjacent,single-stranded DNA oligonucleotides splinted by a complementary RNAstrand. In some instances, the ligase is a single-stranded DNA ligase.In some embodiments, the ligase is a pre-activated T4 DNA ligase.Methods of utilizing a pre-activated T4 DNA ligase are further disclosedin U.S. Publication No. 2010-0184618-A1, which is incorporated byreference in its entirety.

In some embodiments, adenosine triphosphate (ATP) is added during theligation reaction. DNA ligase-catalyzed sealing of nicked DNA substratesis first activated through ATP hydrolysis, resulting in covalentaddition of an AMP group to the enzyme. After binding to a nicked sitein a DNA duplex, the ligase transfers this AMP to the phosphorylated5′-end at the nick, forming a 5′-5′ pyrophosphate bond. Finally, theligase catalyzes an attack on this pyrophosphate bond by the OH group atthe 3′-end of the nick, thereby sealing it, whereafter ligase and AMPare released. If the ligase detaches from the substrate before the 3′attack, e.g. because of premature AMP reloading of the enzyme, then the5′ AMP is left at the 5′-end, blocking further coupling (e.g., ligation)attempts. In some instances, ATP is added at a concentration of about 1μM, about 10 μM, about 100 μM, about 1000 μM, or about 10000 μM duringthe coupling (e.g., ligation) reaction.

In some instances, cofactors that aid in ligating of the probes areadded during the ligation process. In some instances, the cofactorsinclude magnesium ions (Mg²⁺). In some instances, the cofactors includemanganese ions (Mn²⁺). In some instances, Mg²⁺ is added in the form ofMgCl₂. In some instances, Mn²⁺ is added in the form of MnCl₂. In someinstances, the concentration of MgCl₂ is at about 1 mM, at about 10 mM,at about 100 mM, or at about 1000 mM. In some instances, theconcentration of MnCl₂ is at about 1 mM, at about 10 mM, at about 100mM, or at about 1000 mM.

In some instances, the ligation reaction occurs at a pH in the range ofabout 6.5 to 9.0, of about 6.5 to 8.0, of about 7.5 to 8.0, of about7.5, or of about 8.0.

In some instances, a single probe having multiple sequences thathybridize to a nucleic acid is used. For example, in some instances, thefirst probe and the second probe are on a contiguous nucleic acid. Insome instances, the first probe is on the 3′ end of the contiguousnucleic acid and the second probe is on the 5′ end of the contiguousnucleic acid (or vice versa). As described above, in some instances, thetwo sequences of hybridization can be adjacent to one another, or theycan hybridize to sequences that have a gap sequence between the twosites of hybridization. In some instances, a circular nucleic acid canbe formed and amplified (e.g., using rolling circle amplification). Insome instances, the circularized sequence can be digested (e.g., usingan endonuclease), creating a linear nucleic acid that can be capturedand identified using two slide methods disclosed herein. Thus, in someinstances, the single probe includes one or more restriction sites thatis designed to be unique to the probe and is not found in an analyte ofinterest. Any endonuclease known in the art can be used so long as itmeets these criteria.

In some embodiments, methods are provided herein for amplifying thecontiguous nucleic acid, where amplification of the contiguous nucleicacid increases the number of copies of the contiguous nucleic acid. Insome embodiments where a contiguous nucleic acid is amplified, theamplification is performed by rolling circle amplification. In someembodiments, the contiguous nucleic acid to be amplified includessequences (e.g., docking sequences, functional sequences, and/or primersequences) that enable rolling circle amplification. In one example, thecontiguous nucleic acid can include a functional sequence that iscapable of binding to a primer used for amplification. In anotherexample, the contiguous nucleic acid can include one or more dockingsequences (e.g., a first docking sequence and a second docking sequence)that can hybridize to one or more oligonucleotides (e.g., a padlockprobe(s)) used for rolling circle amplification.

As used herein, a “padlock probe” refers to an oligonucleotide that has,at its 5′ and 3′ ends, sequences that are complementary to adjacent ornearby target sequences (e.g., docking sequences) on a target nucleicacid sequence. Upon hybridization to the target sequences (e.g., dockingsequences), the two ends of the padlock probe are either brought intocontact or an end is extended until the two ends are brought intocontact, allowing circularization of the padlock probe by ligation(e.g., ligation using any of the methods described herein). In someembodiments, after circularization of the oligonucleotide, rollingcircle amplification can be used to amplify the ligation product, whichincludes the contiguous nucleic acid.

In some instances, probes (e.g., a first probe and a second probe) areligated with the aid of a splint oligonucleotide. In some instances, thesplint oligonucleotide includes a first splint sequence that issubstantially complementary to the first probe or a portion thereof anda second splint sequence that is substantially complementary to thesecond probe or a portion thereof. The splint oligonucleotide can bebetween 10 and 100 (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99. 100) nucleotides in length. In someinstances, the splint oligonucleotide aids in ligation of the firstprobe and the second probe. Methods including a splint oligonucleotidehave been described in U.S. Patent Pub. No. 2019/0055594A1, which isherein incorporated by reference in its entirety.

In some embodiments, after the step of hybridizing the probes to thenucleic acid, a wash step is performed to remove unbound probes. Thewash step can be performed using any of the wash methods and solutionsdescribed herein. In some embodiments, after the washing step, the firstand second probes are bound to (e.g., hybridized to) the analyte, andthe splint oligonucleotide is bound to (e.g., hybridized to) the firstand second oligonucleotides (e.g., at portions of the first and secondprobes that are not bound to the analyte). In some embodiments, thefirst probe, the second probe, and/or the splint oligonucleotide areadded to the biological sample at the same time. In some embodiments,the first probe and the second probe are added at a first time point,and the splint oligonucleotide is added to the biological sample at asecond time point.

Addition of Methylated Adaptors

Provided herein are methods for identifying a methylation status of DNAin a biological sample, the method comprising: (a) providing one or moremethylated adaptors and a transposase enzyme to the biological sampleunder conditions wherein the one or more methylated adaptors is insertedinto the DNA; (b) contacting the sequence comprising the analyte and theone or more methylated adaptors with an array comprising a plurality ofcapture probes, wherein a capture probe in the plurality of captureprobes comprises a spatial barcode, wherein if the capture probecomprises one or more cytosines, then the one or more cytosines aremethylated cytosines; (c) ligating the sequence comprising the analyteand the one or more methylated adaptors to the capture probe, therebycreating a ligation product; (d) deaminating the ligation product; (e)determining (i) all or a portion of the sequence of the spatial barcodeor the complement thereof, and (ii) all or a portion of the sequence ofthe ligation product, or a complement thereof, and using the determinedsequences of (i) and (ii) to identify the methylation status of theanalyte in the biological sample.

In some embodiments, the method further comprises allowing thetransposase enzyme to fragment the DNA and insert the methylatedadaptors, thereby generating a sequence comprising an analyte and theone or more methylated adaptors, prior to step (b).

Also provided herein are methods for identifying a methylation status ofa nucleic acid in a biological sample on a spatial array, the methodcomprising: (a) ligating methylated adaptors onto the 5′ and/or 3′ endsof fragmented nucleic acids, thereby generating an adapted nucleic acidfragment; (b) deaminating the adapted nucleic acid fragment; (c)capturing the adapted nucleic acid fragment onto a spatial arraycomprising a plurality of capture probes, wherein a capture probescomprise a spatial barcode; (d) determining (i) all or a portion of thesequence of the spatial barcode or the complement thereof, and (ii) allor a portion of the sequence of the methylated adaptor/nucleic acid, ora complement thereof, and using the determined sequences of (i) and (ii)to identify the methylation status of the analyte, or a portion thereof,in the biological sample.

In some instances, a methylated adaptor is ligated to a nucleic acid ata 5′ end of the nucleic acid. In some instances, a methylated adaptor isligated to a nucleic acid at a 3′ end of the nucleic acid. In someinstances, methylated adaptors are ligated to a nucleic acid at the 5′end and the 3′ end of the nucleic acid. In some embodiments, themethylated adaptor is a methylated capture handle.

In some instances, a methylated adaptor is from 10 to 75 (e.g., 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, or 75) nucleotides in length. Insome instances, the methylated adaptor is single-stranded. In someinstances, the methylated adaptor is double-stranded. In some instances,the methylated adaptor is a DNA molecule.

In some instances, the methylated adaptor includes one or moremethylated cytosine, which is protected from mutation caused duringdeamination. For instances, in some embodiments, the methylated adaptorincludes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more methylatedcytosines.

In some instances, the methylated adaptor is ligated to nucleic acids bytagmentation.

In some embodiments, step (a) further comprises allowing a transposaseenzyme to fragment the analytes, e.g., nucleic acids, and insert themethylated adaptors, thereby generating one or more sequences, eachcomprising a nucleic acid analyte and the one or more methylatedadaptors (i.e., an adapted (e.g., tagmented) nucleic acid fragment).

A method for spatial transcriptome analysis using a transposase enzymeis described in U.S. Patent Publication No. WO 2020/047002, which isincorporated herein by reference in its entirety.

As used herein, “tagmentation” refers to a process oftransposase-mediated fragmentation and tagging of DNA. Tagmentationtypically involves the modification of DNA by a transposome complex andresults in the formation of “tagments”, or tagged DNA fragments.

As used herein, a “transposome” or “transposome complex” is a complex ofa transposase enzyme and DNA which comprises transposon end sequences(also known as “transposase recognition sequences” or “mosaic ends”(ME)).

A “transposase” is an enzyme that binds to the end of a transposon andcatalyzes its movement to another part of the genome by a cut and pastemechanism or a replicative transposition mechanism.

The DNA that forms a complex with a transposase enzyme (i.e. thetransposon or ME sequences) contains a partially double stranded (e.g.DNA) oligonucleotide, wherein each strand contains a transposasespecific sequence which forms the double stranded part of theoligonucleotide. The single-stranded portion of the oligonucleotide isat the 5′ end of the oligonucleotide (i.e. forms a 5′ overhang) and maycomprise a functional sequence (e.g. a capture probe binding site, anadaptor sequence, etc.). Thus, the partially double strandedoligonucleotides in the transposome may be viewed as adaptors that canbe ligated to the fragmented DNA. A transposome comprises a transposaseenzyme complexed with one or more adaptors comprising transposon endsequences (or mosaic ends) and tagmentation results in the simultaneousfragmentation of DNA and ligation (e.g., tagging) of the adapters to the5′ ends of both ends of DNA duplex fragments. In some embodiments, anadaptor is methylated (e.g., comprising one or more methylatedcytosines).

It will be evident that tagmentation can be used to provide fragmentedDNA with a domain capable of hybridizing and/or ligating to the capturedomain of the capture probes of the invention. Moreover, the domain maybe provided directly or indirectly.

For example, in some embodiments, the adaptors of the transposomecomprise a functional domain or sequence that may be configured tohybridize to all or a portion of a capture domain. The functional domainor sequence may be a domain capable of hybridizing to the capture domainof the capture probes of the invention (e.g. a homopolymeric sequence,e.g. poly-A sequence). In other words, the single-stranded portion of anadaptor comprises a domain capable of hybridizing to the capture domainof the capture probes of the invention. Accordingly, tagmentationresults in the fragmentation of DNA of the biological specimen andligation of the domain capable of hybridizing to the capture domain ofthe capture probes of the invention to the DNA of the biologicalspecimen, i.e. providing the DNA of the biological specimen with adomain complementary to the capture probe capture domain directly.

In one embodiment, the functional domain or sequence is configured toattach to a portion of the capture domain through click chemistry. Asused herein, the term “click chemistry,” generally refers to reactionsthat are modular, wide in scope, give high yields, generate onlyinoffensive byproducts, such as those that can be removed bynonchromatographic methods, and are stereospecific (but not necessarilyenantioselective). See, e.g., Angew. Chem. Int. Ed., 2001,40(11):2004-2021, which is incorporated herein by reference in itsentirety. In some cases, click chemistry can describe pairs offunctional groups that can selectively react with each other in mild,aqueous conditions.

In some embodiments, an adaptor of a transposome comprises (i) a domaincapable of (i.e. suitable for) facilitating the introduction of a clickchemistry moiety(ies) configured to interact with another clickchemistry moiety(ies) which can be associated with the capture domain ofthe capture probes of the invention.

An example of a click chemistry reaction can be the Huisgen 1,3-dipolarcycloaddition of an azide and an alkyne, i.e., Copper-catalyzed reactionof an azide with an alkyne to form a 5-membered heteroatom ring called1,2,3-triazole. The reaction can also be known as a Cu(I)-CatalyzedAzide-Alkyne Cycloaddition (CuAAC), a Cu(I) click chemistry or a Cu+click chemistry. Catalyst for the click chemistry can be Cu(I) salts, orCu(I) salts made in situ by reducing Cu(II) reagent to Cu(I) reagentwith a reducing reagent (Pharm Res. 2008, 25(10): 2216-2230). KnownCu(II) reagents for the click chemistry can include, but are not limitedto, Cu(II)-(TBTA) complex and Cu(II) (THPTA) complex. TBTA, which istris-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, also known astris-(benzyltriazolylmethyl)amine, can be a stabilizing ligand for Cu(I)salts. THPTA, which is tris-(hydroxypropyltriazolylmethyl)amine, can beanother example of stabilizing agent for Cu(I). Other conditions canalso be accomplished to construct the 1,2,3-triazole ring from an azideand an alkyne using copper-free click chemistry, such as by theStrain-promoted Azide-Alkyne Click chemistry reaction (SPAAC, see, e.g.,Chem. Commun., 2011, 47:6257-6259 and Nature, 2015, 519(7544):486-90),each of which is incorporated herein by reference in its entirety.

In another embodiment, an adaptor of a transposome may comprise anucleotide sequence that templates the ligation of a universal adaptorto the tagmented DNA. The universal adaptor comprises a domain capableof hybridizing to the capture domain of the capture probes of theinvention. Thus, in some embodiments, tagmentation provides the DNA ofthe biological specimen with a domain capable of hybridizing with thecapture domain of a capture probe indirectly.

In another embodiment, an adaptor of a transposome may comprise anucleotide sequence that is a substrate in a ligation reaction thatintroduces a universal adaptor to the tagmented DNA, e.g. a domain towhich a universal adaptor may bind. For instance, the universal adaptormay be a partially double-stranded oligonucleotide having a first strandcomprising a single-stranded portion containing domain that binds to theadaptor sequence ligated to the fragmented (i.e. tagmented) DNA and asecond strand comprising a domain that binds to the first strand and adomain capable of binding (hybridizing) to the capture domain of thecapture probes of the invention. Ligation of the universal adaptor tothe fragmented (i.e. tagmented) DNA provides the tagmented DNA with adomain that binds to the capture domain of the capture probes of theinvention. Thus, in some embodiments, tagmentation provides the DNA ofthe biological specimen with a binding domain indirectly.

As tagmentation results in DNA that comprises gaps between the 3′ endsof the DNA of the biological specimen and the 5′ ends at the doublestranded portion of the adaptors (i.e. the 5′ ends of the adaptorscontaining the MEs are not ligated to the 3′ ends of the fragmented DNAof the biological specimen), providing the tagmented DNA with a bindingdomain capable of binding (hybridizing) to the capture domain of thecapture probes of the invention may require a step of “gap filling” thetagmented DNA.

Gap filling may be achieved using a suitable polymerase enzyme, i.e. aDNA polymerase (e.g. selected from the list below). In this respect, the3′ ends of the tagmented DNA are extended using the complementarystrands of the tagmented DNA as templates. Once the gaps have beenfilled, the 3′ ends of the tagmented DNA are joined to the 5′ ends ofthe adaptors by a ligation step, using a suitable ligase enzyme.

It will be understood in this regard that the 5′ end of adaptorscontaining the ME is phosphorylated to enable ligation to take place.The transposome may comprise an adaptor in which one or both 5′ ends arephosphorylated. In embodiments where the transposome comprises anadaptor in which the 5′ end of adaptor containing the ME is notphosphorylated, the gap filling process may comprise a further step ofphosphorylating the 5′ end of the adaptor, e.g. using a kinase enzyme,such as T4 polynucleotide kinase.

In some embodiments, the 3′ ends of the tagmented DNA may be extendedusing a DNA polymerase with strand displacement activity using thecomplementary strands of the tagmented DNA as templates. This results inthe displacement of the strands of the adaptors that are not ligated tothe fragmented DNA and the generation of fully double stranded DNAmolecules. These molecules may be provided with a domain capable ofbinding to the capture domain of the capture probes by any suitablemeans, e.g. ligation of adaptors, “tailing” with a terminal transferaseenzyme etc.

Thus, in some embodiments, the method comprises a step of extending the3′ ends of the fragmented (i.e. tagmented) DNA using a polymerase withstrand displacement activity to produce fully double stranded DNAmolecules.

In some embodiments, the fully double stranded DNA molecules may beprovided with a binding domain capable of binding to the capture domainof the capture probes on a spatial array. In some embodiments, a bindingdomain may be provided by ligation of adaptors to the double strandedDNA molecules or via the use of a terminal transferase active enzyme toincorporate a polynucleotide tail, e.g. homopolymeric sequence (e.g. apoly-A tail), at the 3′ ends of the double stranded DNA molecules.

Transposase Tn5 is a member of the RNase superfamily of proteins. TheTn5 transposon is a composite transposon in which two near-identicalinsertion sequences (IS50L and IS50R) flank three antibiotic resistancegenes. Each IS50 contains two inverted 19-bp end sequences (ESs), anoutside end (OE) and an inside end (IE).

A hyperactive variant of the Tn5 transposase is capable of mediating thefragmentation of double-stranded DNA and ligation of syntheticoligonucleotides (adaptors) at both 5′ ends of the DNA in a reactionthat takes about 5 minutes. However, as wild-type end sequences have arelatively low activity, they are preferably replaced in vitro byhyperactive mosaic end (ME) sequences. A complex of the Tn5 transposasewith 19-bp ME is thus all that is necessary for transposition to occur,provided that the intervening DNA is long enough to bring two of thesesequences close together to form an active Tn5 transposase homodimer.

Methods, compositions, and kits for treating nucleic acid, and inparticular, methods and compositions for fragmenting and tagging DNAusing transposon compositions are described in detail in US2010/0120098and US2011/0287435, which are hereby incorporated by reference in theirentireties.

Thus, any transposase enzyme with tagmentation activity, i.e. capable offragmenting DNA and ligating oligonucleotides to the ends of thefragmented DNA, may be used in the methods of the present invention. Insome embodiments, the transposase is a Tn5, Tn7, or Mu transposase or afunctional variant or derivative thereof.

The tagmentation of the nucleic acid in the biological sample can beperformed before or after the deamination of the nucleic acid. In someembodiments, the nucleic acid is deaminated prior to the tagmentation.In some embodiments, the nucleic acid is deaminated after thetagmentation.

In some embodiments, the method further comprises washing the biologicalsample. In some instances, a wash step occurs before or after thedeamination step. In some embodiments, the washing step is conducted toremove all or a part of the deaminating agent. In some embodiments, thewashing step does not remove the analyte in the biological sample. Insome embodiments, the washing step removes an insignificant amount ofthe analyte in the biological sample. In some instances, a wash stepoccurs after hybridizing the oligonucleotides. In some instances, thiswash step removes any unbound oligonucleotides and can be performedusing any technique or solution disclosed herein or known in the art. Insome embodiments, multiple wash steps are performed to remove unboundoligonucleotides.

Two-Slide Method Processes

In some embodiments, the alignment of the first substrate and the secondsubstrate is facilitated by a sandwiching process. Accordingly,described herein are methods of positioning together the first substrateas described herein with a second substrate having an array with captureprobes.

FIG. 12 is a schematic diagram depicting an exemplary sandwichingprocess 1201 between a first substrate comprising a biological sample(e.g., a tissue section 1202 on a pathology slide 1203) and a secondsubstrate comprising a spatially barcoded array, e.g., a slide 1204 thatis populated with spatially-barcoded capture probes 1206. During theexemplary sandwiching process, the first substrate is aligned with thesecond substrate, such that at least a portion of the biological sampleis aligned with at least a portion of the array (e.g., aligned with thearray area superior or inferior to the biological sample). As shown, thearrayed slide 1204 is in a superior position to the pathology slide1203. In some embodiments, the pathology slide 1203 may be positionedsuperior to the arrayed slide 1204. In some embodiments, the first andsecond substrates are aligned to maintain a gap or separation distance1207 between the two substrates. When the first and second substratesare aligned, one or more analytes are released from the biologicalsample and actively or passively migrated to the array for capture. Insome embodiments, the migration occurs while the aligned portions of thebiological sample and the array are contacted with a reagent medium1205. The released one or more analytes may actively or passivelymigrate across the gap 1207 via the reagent medium 1205 toward thecapture probes 1206, and be captured by the capture probes 1206.

In some embodiments, the separation distance in the gap 1207 betweenfirst and second substrates is maintained between 2 microns and 1 mm(e.g., between 2 microns and 800 microns, between 2 microns and 700microns, between 2 microns and 600 microns, between 2 microns and 500microns, between 2 microns and 400 microns, between 2 microns and 300microns, between 2 microns and 200 microns, between 2 microns and 100microns, between 2 microns and 25 microns, between 2 microns and 10microns), measured in a direction orthogonal to the surface of firstsubstrate that supports sample. In some instances, the distance is 2microns. In some instances, the distance is 2.5 microns. In someinstances, the distance is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 microns. In someembodiments, second substrate is placed in direct contact with thesample on the first substrate ensuring no diffusive spatial resolutionlosses. In some embodiments, the separation distance is measured in adirection orthogonal to a surface of the first substrate that supportsthe biological sample.

In some embodiments, the first and second substrates are placed in asubstrate holder (e.g., an array alignment device) configured to alignthe biological sample and the array. In some embodiments, the devicecomprises a sample holder. In some embodiments, the sample holderincludes a first and second member that receive a first and secondsubstrate, respectively. The device can include an alignment mechanismthat is connected to at least one of the members and aligns the firstand second members. Thus, the devices of the disclosure canadvantageously align the first substrate and the second substrate andany samples, barcoded probes, or permeabilization reagents that may beon the surface of the first and second substrates. Exemplary devices andexemplary sample holders are described in PCT Patent ApplicationPublication No. WO 2020/123320, which is incorporated by reference inits entirety.

In some embodiments, the reagent medium comprises a permeabilizationagent. Suitable agents for this purpose include, but are not limited to,organic solvents (e.g., acetone, ethanol, and methanol), cross-linkingagents (e.g., paraformaldehyde), detergents (e.g., saponin, TritonX-100™, Tween-20™, or sodium dodecyl sulfate (SDS)), and enzymes (e.g.,trypsin, proteases (e.g., proteinase K). In some embodiments, thedetergent is an anionic detergent (e.g., SDS or N-lauroylsarcosinesodium salt solution). Exemplary permeabilization reagents are describedin in PCT Patent Application Publication No. WO 2020/123320, which isincorporated by reference in its entirety.

In some embodiments, the reagent medium comprises a lysis reagent. Lysissolutions can include ionic surfactants such as, for example, sarkosyland sodium dodecyl sulfate (SDS). More generally, chemical lysis agentscan include, without limitation, organic solvents, chelating agents,detergents, surfactants, and chaotropic agents. Exemplary lysis reagentsare described in PCT Patent Application Publication No. WO 2020/123320,which is incorporated by reference in its entirety.

In some embodiments, the reagent medium comprises a protease. Exemplaryproteases include, e.g., pepsin, trypsin, pepsin, elastase, andproteinase K. Exemplary proteases are described in PCT PatentApplication Publication No. WO 2020/123320, which is incorporated byreference in its entirety.

In some embodiments, the reagent medium comprises a detergent. Exemplarydetergents include sodium dodecyl sulfate (SDS), sarkosyl, saponin,Triton X-100™, and Tween-20™. Exemplary detergents are described in PCTPatent Application Publication No. WO 2020/123320, which is incorporatedby reference in its entirety.

In some embodiments, the reagent medium comprises a nuclease. In someembodiments, the nuclease comprises an RNase. In some embodiments, theRNase is selected from RNase A, RNase C, RNase H, and RNase I. In someembodiments, the reagent medium comprises one or more of sodium dodecylsulfate (SDS), proteinase K, pepsin, N-lauroylsarcosine, RNAse, and asodium salt thereof.

The sample holder is compatible with a variety of different schemes forcontacting the aligned portions of the biological sample and array withthe reagent medium to promote analyte capture. In some embodiments, thereagent medium is deposited directly on the second substrate (e.g.,forming a reagent medium that includes the permeabilization reagent andthe feature array), and/or directly on the first substrate. In someembodiments, the reagent medium is deposited on the first and/or secondsubstrate, and then the first and second substrates aligned in thedisclosed configuration such that the reagent medium contacts thealigned portions of the biological sample and array. In someembodiments, the reagent medium is introduced into the gap 1207 whilethe first and second substrates are aligned in the disclosedconfiguration.

In certain embodiments a dried permeabilization reagent is applied orformed as a layer on the first substrate or the second substrate or bothprior to contacting the sample and the feature array. For example, areagent can be deposited in solution on the first substrate or thesecond substrate or both and then dried. Drying methods include, but arenot limited to spin coating a thin solution of the reagent and thenevaporating a solvent included in the reagent or the reagent itself.Alternatively, in other embodiments, the reagent can be applied in driedform directly onto the first substrate or the second substrate or both.In some embodiments, the coating process can be done in advance of theanalytical workflow and the first substrate and the second substrate canbe stored pre-coated. Alternatively, the coating process can be done aspart of the analytical workflow. In some embodiments, the reagent is apermeabilization reagent. In some embodiments, the reagent is apermeabilization enzyme, a buffer, a detergent, or any combinationthereof. In some embodiments, the permeabilization enzyme is pepsin. Insome embodiments, the reagent is a dried reagent (e.g., a reagent freefrom moisture or liquid). In some instances, the substrate that includesthe sample (e.g., a histological tissue section) is hydrated. The samplecan be hydrated by contacting the sample with a reagent medium, e.g., abuffer that does not include a permeabilization reagent. In someembodiments, the hydration is performed while the first and secondsubstrates are aligned in a sandwich style configuration.

In some instances, the aligned portions of the biological sample and thearray are in contact with the reagent medium 1205 in the gap 1207 forabout 1 minute. In some instances, the aligned portions of thebiological sample and the array are in contact with the reagent medium1205 for about 5 minutes. In some instances, the aligned portions of thebiological sample and the array are in contact with the reagent medium1205 in the gap 1207 for about 1 minute, about 5 minutes, about 10minutes, about 12 minutes, about 15 minutes, about 18 minutes, about 20minutes, about 25 minutes, about 30 minutes, about 36 minutes, about 45minutes, or about an hour. In some instances, the aligned portions ofthe biological sample and the array are in contact with the reagentmedium 1205 for about 1-60 minutes. In some instances, the alignedportions of the biological sample and the array are in contact with thereagent medium 1205 for about 30 minutes.

In some embodiments, following initial contact between a sample and apermeabilization agent, the permeabilization agent can be removed fromcontact with the sample (e.g., by opening the sample holder) beforecomplete permeabilization of the sample. For example, in someembodiments, only a portion of the sample is permeabilized, and only aportion of the analytes in the sample may be captured by the featurearray. In some instances, the reduced amount of analyte captured andavailable for detection can be offset by the reduction in lateraldiffusion that results from incomplete permeabilization of the sample.In general, the spatial resolution of the assay is determined by theextent of analyte diffusion in the transverse direction (i.e.,orthogonal to the normal direction to the surface of the sample). Thelarger the distance between the sample on the first substrate and thefeature array on the second substrate, the greater the extent ofdiffusion in the transverse direction, and the concomitant loss ofresolution. Analytes liberated from a portion of the sample closest tothe feature array have a shorter diffusion path, and therefore do notdiffuse as far laterally as analytes from portions of the samplefarthest from the feature array. As a result, in some instances,incomplete permeabilization of the sample (by reducing the contactinterval between the permeabilization agent and the sample) can be usedto maintain adequate spatial resolution in the assay.

In some instances, the device is configured to control a temperature ofthe first and second substrates. In some embodiments, the temperature ofthe first and second members is lowered to a first temperature that isbelow room temperature (e.g., 25 degrees Celsius) (e.g., 20 degreesCelsius or lower, 15 degrees Celsius or lower, 10 degrees Celsius orlower, 5 degrees Celsius or lower, 4 degrees Celsius or lower, 3 degreesCelsius or lower, 2 degrees Celsius or lower, 1 degree Celsius or lower,0 degrees Celsius or lower, −1 degrees Celsius or lower, −5 degreesCelsius or lower). In some embodiments, the device includes atemperature control system (e.g., heating and cooling conducting coils)to control the temperature of the sample holder. Alternatively, in otherembodiments, the temperature of the sample holder is controlledexternally (e.g., via refrigeration or a hotplate). In a first step, thesecond member, set to or at the first temperature, contacts the firstsubstrate, and the first member, set to or at the first temperature,contacts the second substrate, thereby lowering the temperature of thefirst substrate and the second substrate to a second temperature. Insome embodiments, the second temperature is equivalent to the firsttemperature. In some embodiments, the first temperature is lower thanroom temperature (e.g., 25 degrees Celsius). In some embodiments, thesecond temperature ranges from about −10 degrees Celsius to about 4degrees Celsius. In some embodiments, the second temperature is belowroom temperature (e.g., 25 degrees Celsius) (e.g., 20 degrees Celsius orlower, 15 degrees Celsius or lower, 10 degrees Celsius or lower, 5degrees Celsius or lower, 4 degrees Celsius or lower, 3 degrees Celsiusor lower, 2 degrees Celsius or lower, 1 degree Celsius or lower, 0degrees Celsius or lower, −1 degrees Celsius or lower, −5 degreesCelsius or lower).

In an exemplary embodiment, the second substrate is contacted with thepermeabilization reagent. In some embodiments, the permeabilizationreagent is dried. In some embodiments, the permeabilization reagent is agel or a liquid. Also in the exemplary embodiments, the biologicalsample is contacted with buffer. Both the first and second substratesare placed at lower temperature to slow down diffusion andpermeabilization efficiency. Alternatively, in some embodiments, thesample can be contacted directly with a liquid permeabilization reagentwithout inducing an unwanted initiation of permeabilization due to thesubstrates being at the second temperature. In some embodiments, the lowtemperature slows down or prevents the initiation of permeabilization.In a second step, keeping the sample holder and substrates at a coldtemperature (e.g., at the first or second temperatures) continues toslow down or prevent the permeabilization of the sample. In a thirdstep, the sample holder (and consequently the first and secondsubstrates) is heated up to initiate permeabilization. In someembodiments, the sample holder is heated up to a third temperature. Insome embodiments, the third temperature is above room temperature (e.g.,25 degrees Celsius) (e.g., 30 degrees Celsius or higher, 35 degreesCelsius or higher, 40 degrees Celsius or higher, 50 degrees Celsius orhigher, 60 degrees Celsius or higher). In some embodiments, analytesthat are released from the permeabilized tissue of the sample diffuse tothe surface of the second substrate and are captured on the array (e.g.,barcoded probes) of the second substrate. In a fourth step, the firstsubstrate and the second substrate are separated (e.g., pulled apart)and temperature control is stopped.

In some embodiments, where either the first substrate or secondsubstrate (or both) includes wells, a permeabilization solution can beintroduced into some or all of the wells, and the sample and the featurearray can be contacted by closing the sample holder to permeabilize thesample. In certain embodiments, a permeabilization solution can besoaked into a hydrogel film that is applied directly to the sample,and/or soaked into features (e.g., beads) of the array. When the firstand second substrates are aligned in the sandwich type configuration,the permeabilization solution promotes migration of analytes from thesample to the array.

In certain embodiments, different permeabilization agents or differentconcentrations of permeabilization agents can be infused into arrayfeatures (e.g., beads) or into a hydrogel layer as described above. Bylocally varying the nature of the permeabilization reagent(s), theprocess of analyte capture from the sample can be spatially adjusted.

In some instances, migration of the analyte from the biological sampleto the second substrate is passive (e.g., via diffusion). Alternatively,in certain embodiments, migration of the analyte from the biologicalsample is performed actively (e.g., electrophoretic, by applying anelectric field to promote migration). In some instances, first andsecond substrates can include a conductive epoxy. Electrical wires froma power supply can connect to the conductive epoxy, thereby allowing auser to apply a current and generate an electric field between the firstand second substrates. In some embodiments, electrophoretic migrationresults in higher analyte capture efficiency and better spatial fidelityof captured analytes (e.g., on a feature array) than random diffusiononto matched substrates without the application of an electric field(e.g., via manual alignment of the two substrates). Exemplary methods ofelectrophoretic migration, including those illustrated in FIGS. 6-9 ,are described in WO 2020/176788, including at FIGS. 14A-14B, 15,24A-24B, and 25A-25C, which is hereby incorporated by reference in itsentirety.

Loss of spatial resolution can occur when analytes migrate from thesample to the feature array and a component of diffusive migrationoccurs in the transverse (e.g., lateral) direction, approximatelyparallel to the surface of the first substrate on which the sample ismounted. To address this loss of resolution, in some embodiments, apermeabilization agent deposited on or infused into a material withanisotropic diffusion can be applied to the sample or to the featurearray. The first and second substrates are aligned by the sample holderand brought into contact. A permeabilization layer that includes apermeabilization solution infused into an anisotropic material ispositioned on the second substrate.

In some embodiments, the feature array can be constructed atop ahydrogel layer infused with a permeabilization agent. The hydrogel layercan be mounted on the second substrate, or alternatively, the hydrogellayer itself may function as the second substrate. When the first andsecond substrates are aligned, the permeabilization agent diffuses outof the hydrogel layer and through or around the feature array to reachthe sample. Analytes from the sample migrate to the feature array.Direct contact between the feature array and the sample helps to reducelateral diffusion of the analytes, mitigating spatial resolution lossthat would occur if the diffusive path of the analytes was longer.

Spatial analysis workflows can include a sandwiching type processdescribed herein, e.g., a process as described in FIG. 12 . In someembodiments, the workflow includes provision of the first substratecomprising the biological sample. In some embodiments, the workflowincludes, mounting the biological sample onto the first substrate. Insome embodiments wherein the biological sample is a tissue sample, theworkflow includes sectioning of the tissue sample (e.g., cryostatsectioning). In some embodiments, the workflow includes a fixation step.In some instances, the fixation step can include fixation with methanol.In some instances, the fixation step includes formalin (e.g., 2%formalin).

In some embodiments, the biological sample on the first substrate isstained using any of the methods described herein. In some instances,the biological sample is imaged, capturing the stain pattern createdduring the stain step. In some instances, the biological sample then isdestained prior to the two slide process 1201.

The biological sample can be stained using known staining techniques,including, without limitation, Can-Grunwald, Giemsa, hematoxylin andeosin (H&E), hematoxylin, Jenner's, Leishman, Masson's trichrome,Papanicolaou, Romanowsky, silver, Sudan, Wright's, and/or Periodic AcidSchiff (PAS) staining techniques. PAS staining is typically performedafter formalin or acetone fixation. In some embodiments, the biologicalsample can be stained using a detectable label (e.g., radioisotopes,fluorophores, chemiluminescent compounds, bioluminescent compounds, anddyes) as described elsewhere herein. In some embodiments, a biologicalsample is stained using only one type of stain or one technique. In someembodiments, staining includes biological staining techniques such asH&E staining. In some embodiments, staining includes biological stainingusing hematoxylin. In some embodiments, staining includes identifyinganalytes using fluorescently-conjugated antibodies, e.g., byimmunofluorescence. In some embodiments, a biological sample is stainedusing two or more different types of stains, or two or more differentstaining techniques. For example, a biological sample can be prepared bystaining and imaging using one technique (e.g., H&E staining andbrightfield imaging), followed by staining and imaging using anothertechnique (e.g., IHC/IF staining and fluorescence microscopy) for thesame biological sample. In some instances, a biological sample on thefirst substrate is stained.

In some instances, methods for immunofluorescence include a blockingstep. The blocking step can include the use of blocking probes todecrease nonspecific binding of the antibodies. The blocking step canoptionally further include contacting the biological sample with adetergent. In some instances, the detergent can include Triton X-100™.The method can further include an antibody incubation step. In someembodiments, the antibody incubation step effects selective binding ofthe antibody to antigens of interest in the biological sample. In someembodiments, the antibody is conjugated to an oligonucleotide (e.g., anoligonucleotide-antibody conjugate as described herein). In someembodiments, the antibody is not conjugated to an oligonucleotide. Insome embodiments, the method further comprises an antibody stainingstep. The antibody staining step can include a direct method ofimmunostaining in which a labelled antibody binds directly to theanalyte being stained for. Alternatively, the antibody staining step caninclude an indirect method of immunostaining in which a first antibodybinds to the analyte being stained for, and a second, labelled antibodybinds to the first antibody. In some embodiments, the antibody stainingstep is performed prior to the two-slide spatial array assembly. In someembodiments wherein an oligonucleotide-antibody conjugate is used in theantibody incubation step, the method does not comprise an antibodystaining step.

In some instances, the methods include imaging the biological sample. Insome instances, imaging occurs prior to a two-slide assembly. In someinstances, imaging occurs while the two-slide configuration isassembled. In some instances, imaging occurs during permeabilization ofthe biological sample. In some instances, image are captured using highresolution techniques (e.g., having 300 dots per square inch (dpi) orgreater). For example, images can be captured using brightfield imaging(e.g., in the setting of hematoxylin or H&E stain), or usingfluorescence microscopy to detect adhered labels. In some instances,high resolution images are captured temporally using e.g., confocalmicroscopy. In some instances, a low resolution image is captured. A lowresolution image (e.g., images that are about 72 dpi and normally havean RGB color setting) can be captured at any point of the workflow,including but not limited to staining, destaining, permeabilization,two-slide assembly, and migration of the analytes. In some instances, alow resolution image is taken during permeabilization of the biologicalsample.

In some embodiments, the location of the one or more analytes in abiological sample are determined by immunofluorescence. In someembodiments, one or more detectable labels (e.g., fluorophore-labeledantibodies) bind to the one or more analytes that are captured(hybridized to) by a probe on the first slide and the location of theone or more analytes is determined by detecting the labels undersuitable conditions. In some embodiments, one or morefluorophore-labeled antibodies are used to conjugate to a moiety thatassociates with a probe on the first slide or the analyte that ishybridized to the probe on the first slide. In some instances, thelocation(s) of the one or more analytes is determined by imaging thefluorophore-labeled antibodies when the fluorophores are excited by alight of a suitable wavelength. In some embodiments, the location of theone or more analytes in the biological sample is determined bycorrelating the immunofluorescence data to an image of the biologicalsample. In some instances, the tissue is imaged throughout thepermeabilization step.

In some instances, the biological samples can be destained. In someinstances, destaining occurs prior to permeabilization of the biologicalsample. By way of example only, H&E staining can be destained by washingthe sample in HCl. In some instances, the hematoxylin of the H&E stainis destained by washing the sample in HCl. In some embodiments,destaining can include 1, 2, 3, or more washes in HCl. In someembodiments, destaining can include adding HCl to a downstream solution(e.g., permeabilization solution).

Between any of the methods disclosed herein, the methods can include awash step (e.g., with SSC (e.g., 0.1×SSC)). Wash steps can be performedonce or multiple times (e.g., 1×, 2×, 3×, between steps disclosedherein). In some instances, wash steps are performed for about 10seconds, about 15 seconds, about 20 seconds, about 30 seconds, or abouta minute. In some instances, three washes occur for 20 seconds each. Insome instances, the wash step occurs before staining the sample, afterdestaining the sample, before permeabilization the sample, afterpermeabilization the sample, or any combination thereof.

In some instances, after the sandwiching type process 1201 the firstsubstrate and the second substrate are separated (e.g., such that theyare no longer aligned in a sandwich type configuration). In someembodiments, subsequent analysis (e.g., cDNA synthesis, librarypreparation, and sequences) can be performed on the captured analytesafter the first substrate and the second substrate are separated.

In some embodiments, the process of transferring the ligation product ormethylated-adaptor-containing nucleic acid from the first substrate tothe second substrate is referred to interchangeably herein as a“sandwich type or sandwich style process,” or “two-slide process”. Thetwo-slide process is further described in PCT Patent ApplicationPublication No. WO 2020/123320, which is incorporated by reference inits entirety.

Release of Deaminated Nucleic Acid, Capture on an Array, andDetermination of Methylation Status

In some embodiments, the methods described herein further comprisereleasing the ligation product from the analyte, e.g., nucleic acid, onthe first substrate and is captured on a second substrate using thesandwich type methods described herein. In some embodiments, thereleasing occurs before capturing of the ligation product to the captureprobe on the spatial array.

In some instances, the ligation product is dehybridized from the analyteby heating the sample. In some instances, the ligation product isdehybridized at about 50° C., about 55° C., about 60° C., about 65° C.,about 70° C., or about 75° C. In some instances, the ligation product isdehybridized from the analyte electrochemically. In some instances, theligation product is dehybridized using a solution comprising a base. Insome instances, the base is potassium hydroxide (KOH). In someinstances, the base is sodium hydroxide (NaOH). In some instances, torestore neutral pH after this step, an acid (e.g., a weak acid) having apH below 7 (e.g., pH of 5, 5.5, 6, 6.5) can be added.

As described above, after release of the ligation product, it canmigrate (actively or passively) to the second substrate and can becaptured on the array on the second substrate.

In some instances, the second substrate comprises an array. In someinstances, the array includes a plurality of capture probes, each ofwhich includes a capture domain and a spatial barcode. In someembodiments, the capture probe is affixed to the substrate at a 5′ end.In some embodiments, the plurality of capture probes are uniformlydistributed on a surface of the substrate. In some embodiments, theplurality of capture probes are located on a surface of the substratebut are not distributed on the substrate according to a pattern. In someembodiments, the substrate (e.g., a second substrate) includes aplurality of capture probes, where a capture probe of the plurality ofcapture probes includes a capture domain and a spatial barcode.

In some embodiments, the capture domain includes a sequence that is atleast partially complementary to the analyte or the analyte derivedmolecule. In some embodiments, the capture domain of the capture probeincludes a poly(T) sequence. In some embodiments, the capture domainincludes a functional domain. In some embodiments, the functional domainincludes a primer sequence. In some embodiments, the capture probeincludes a cleavage domain. In some embodiments, the cleavage domainincludes a cleavable linker from the group consisting of aphotocleavable linker, a UV-cleavable linker, an enzyme-cleavablelinker, or a pH-sensitive cleavable linker.

The array can be any suitable array described herein. In someembodiments, the array comprises a slide. In some embodiments, the arrayincludes a plurality of capture probes. In some embodiments, a 5′ end ofa capture probe in the plurality is attached to the slide. In someembodiments, the array is a bead array. In some embodiments, a 5′ end ofthe capture probe is attached to a bead of the bead array. In someembodiments, the capture probe further comprises a unique molecularidentifier (UMI). The UMI can be any suitable UMI described herein. Insome embodiments, the UMI is positioned 5′ relative to the capturedomain in the capture probe. In some embodiments, the capture probefurther comprises a functional sequence. In some embodiments, thefunctional sequence is a primer sequence. In some embodiments, theprimer sequence is used to extend the capture probe.

In some embodiments, the capture domain of the capture probe comprises asequence that specifically binds to the overhang sequence of one or moreprobes. The spatial barcode can be any suitable spatial barcodesdescribed herein.

In some embodiments, methods provided herein include contacting abiological sample with a substrate, wherein the capture probe is affixedto the substrate (e.g., immobilized to the substrate, directly orindirectly). In some embodiments, the capture probe includes a spatialbarcode and a capture domain. In some embodiments, the capture probebinding domain of the ligation product specifically binds to the capturedomain. After hybridization of the ligation product to the captureprobe, the ligation product is extended at the 3′ end to make a copy ofthe additional components (e.g., the spatial barcode) of the captureprobe. In some embodiments, methods of ligation product capture asprovided herein include permeabilization of the biological sample suchthat the capture probe can more easily hybridize to the ligation product(i.e., compared to no permeabilization).

In some instances, after the ligation product hybridizes to the captureprobe, the method disclosed herein includes extending the capture probeusing the ligation product as a template; thereby generating an extendedcapture probe. In some instances, the capture probe is extended whereineach adenine, if present in the extended probe, is base-paired with athymine. In some embodiments, the extended capture probe comprises athymine in each position corresponding to the unmethylated cytosines inthe analyte in the biological sample. In some embodiments, themethylated cytosines, e.g., 5-methylcytosine or 5-hydroxymethylcytosine,if present in the analyte in the sample, remain unchanged.

In some embodiments, amplification reagents can be added to secondsubstrate. Incubation with the amplification reagents can producespatially-barcoded DNA sequences. Second strand reagents (e.g., secondstrand primers, enzymes) can be added to the biological sample on theslide to initiate second strand DNA synthesis.

The resulting DNA can be denatured from the capture probe template andtransferred (e.g., to a clean tube) for amplification, and/or libraryconstruction as described herein. The spatially-barcoded, full-lengthDNA can be amplified via PCR prior to library construction. The DNA canthen be enzymatically fragmented and size-selected in order to optimizethe DNA amplicon size. Sequencing specific nucleic acid sequences suchas P5, P7, sample indices such as i7, and i5 and sequencing primersequences such as TruSeq Read 2 (exemplary sequences that are used inIllumina NGS sequencing workflows) can be added via End Repair,A-tailing, Adaptor Ligation, and PCR. The cDNA fragments can then besequenced using paired-end sequencing using TruSeq Read 1 and TruSeqRead 2 as sequencing primer sites. In some instances, the DNA library issequenced using any method described herein.

In some embodiments, the capture probe, ligation product or a complementthereof, and/or analyte/methylated adaptor can be sequenced. In someinstances, this is performed using a determining step. In someinstances, the determining step comprises sequencing (i) all or a partof the sequence of the analyte, or a complement thereof, and (ii) all ora part of the sequence of the spatial barcode, or a complement thereof.The sequencing can be performed using any suitable methods describedherein. In some embodiments, the sequencing is high throughputsequencing. In some embodiments, the sequencing is sequencing byhybridization. In some embodiments, the sequencing is sequencing bysynthesis. In some embodiments, the sequencing is sequencing byligation. In some embodiments, the sequencing is nanopore typesequencing.

In some embodiments, the methods described herein further comprisestransferring an analyte to a spatial array (e.g., a spatial array on asubstrate). In some embodiments, the methods further comprise amigration step wherein the analyte migrates to the substrate. In someembodiments, the migration is an active migration step. For example, themigration is through a microfluidic system or a concentration gradientof a reagent. In some embodiments, active migration is effected byelectrophoretic means. In some embodiments, the migration is a passivemigration step. For example, the migration follows gravity. In someembodiments, the analyte is a ligation product (e.g., a ligated probe).In some embodiments, the analyte is an adapted (e.g., tagmented) nucleicacid fragment. In some embodiments, the adapted (e.g., tagmented)nucleic acid fragment is deaminated.

In some embodiments, identification of the methylated cytosines isindicative of the methylation status of the analyte, e.g., nucleic acid.In some embodiments, the methylation status of the analyte is indicatedby the percentage of methylated cytosines over all cytosines in theanalyte. In some embodiments, the methods described herein furthercomprise comparing the determined sequence of the analyte with areference sequence of the analyte, e.g., the sequence of the analytewithout deamination. In some embodiments, the comparison comprisesidentifying thymines in the determined sequence of the analyte andcomparing with the nucleotides in the corresponding positions in thereference sequence of the analyte, and determining that one or morethymines in the determined sequence using the methods described hereinare unmethylated cytosines. In some embodiments, one or more cytosinesin the analyte, e.g., DNA, can be methylated cytosines. In someembodiments, the determined sequence of the analyte comprises one ormore cytosines, wherein the one or more cytosines are methylatedcytosines. In some embodiments, about 0.0001% to about 50% (e.g., about0.0001% to about 0.001%, about 0.001% to about 0.01%, about 0.01 toabout 0.10%, about 0.1% to about 0.2%, about 0.2% to about 0.3%, about0.3% to about 0.4%, about 0.4% to about 0.5%, about 0.5% to about 0.6%,about 0.6% to about 0.7%, about 0.7% to about 0.8%, about 0.8% to about0.9%, about 0.9% to about 10%, about 10% to about 2%, about 2% to about3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%,about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about9% to about 10%, about 10% to about 15%, about 15% to about 20%, about20% to about 25%, about 25% to about 30%, about 30% to about 35%, about35% to about 40%, about 40% to about 45%, or about 45% to about 50%) ofthe cytosines in an analyte are methylated. In some embodiments, nocytosine in an analyte is methylated.

In some instances, the percentage or abundance of methylated nucleotidescan be determined and converted into an image, such as a heat map asshown in FIG. 11B. In some instances, the map is generated based on thepercentage of methylated nucleotides (e.g., cytosines) or abundance ofmethylated nucleotides (e.g., cytosines) by determining the location ofthe methylated nucleotides (e.g., cytosines) using the location of thespatial barcode in the capture probe as provided by sequencing results.

In some embodiments, the methods provided herein comprise comparing themethylation status of the analyte at a location in the tissue sample tothe DNA methylation status of a reference location in a reference tissuesample. In some embodiments, the reference location is a location in ahealthy tissue, e.g., a healthy tissue corresponding to the tissue wherethe tissue sample is obtained. In some embodiments, the referencelocation is a location in a non-cancerous tissue. In some embodiments,the reference location is in a non-tumor tissue. In some embodiments,the reference location is a location in a tissue sample with noabnormalities such as tumor, cancer, or disease. In some embodiments,the reference location is in a sample identified as having cancer. Insome instances, the reference location is a location that includes atumor.

In some embodiments, the methylation status of the analyte in the tissuesample is different from the methylation status of an analyte in areference location in a reference tissue sample. In some embodiments,the methylation of the analyte in the tissue sample is higher than themethylation of an analyte in a reference location. In some embodiments,the methylation of the analyte in the tissue sample is lower than themethylation of an analyte in a reference location. In some instances,the methylation status of a particular cytosine of an analyte isdifferent from a cytosine in an analyte at a reference location. In someinstances, a methyl group is located at a particular cytosine comparedto a corresponding cytosine in the reference location. In someinstances, a methyl group is not present at a particular cytosinecompared to a methyl group present on a cytosine in the referencelocation. In some instances, the methylation status of the analyteincludes at least 2, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10, at least 20, at least 30,at least 40, at least 50, or more methyl groups compared to thecorresponding cytosines in the reference location. In some instances,the methylation status of the analyte does not include at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 20, at least 30, at least 40, at least50, or more methyl groups compared to the corresponding cytosines in thereference location of a reference tissue sample.

Kits and Compositions

In some embodiments, also provided herein are kits and compositions thatinclude one or more reagents to detect the methylation status of anucleic acid in a biological sample.

In some instances, the kit or composition includes a first substrate asdescribed herein. In some instances, the kit or composition includes asecond substrate as described herein. In some instances, the kit orcomposition include means for releasing one or more nucleic acids and/orprobes from a biological sample and allowing the one or more nucleicacids and/or probes to migrate to the second substrate. In someembodiments, the kit or composition includes a substrate comprising aplurality of capture probes, wherein a capture probe of the plurality offirst capture probes comprises (i) a spatial barcode and (ii) a capturedomain.

In some instances, the kit or composition includes one or more mixtures,reagents, or solutions for deaminating a nucleotide or nucleic acid. Insome instances, the one or more mixtures, reagents, or solutions issodium bisulfite.

In some embodiments, reagents can include one or more antibodies (and/orantigen-binding antibody fragments), labeled hybridization probes, andprimers. For example, in some embodiments, an antibody (and/orantigen-binding antibody fragment) can be used for visualizing one ormore features of a tissue sample (e.g., by using immunofluorescence orimmunohistochemistry). In some embodiments, an antibody (and/orantigen-binding antibody fragment) can be an analyte binding moiety, forexample, as part of an analyte capture agent. For example, in someembodiments, a kit can include an anti-PMCH antibody, such as ProductNo. HPA046055 (Atlas Antibodies), Cat. Nos. PA5-25442, PA5-84521,PA5-83802 (ThermoFisher Scientific), or Product No. AV13054(MilliporeSigma). Other useful commercially available antibodies will beapparent to one skilled in the art.

In some embodiments, labeled hybridization probes can be used for insitu sequencing of one or more biomarkers and/or candidate biomarkers.In some embodiments, primers can be used for amplification (e.g., clonalamplification) of a captured oligonucleotide analyte.

In some embodiments, a kit or composition can include enzyme systems forperforming DNA tagmentation on a target analyte. Enzyme systems caninclude one or more transposases, transposon sequences, adaptorsequences, either separately or combined into transposome complexes. Insome embodiments, the enzyme is selected from the group consisting of atransposase, a ligase, a polymerase; a reverse transcriptase, a cytidinedeaminase and a demethylase.

In some embodiments, a kit or composition can further includeinstructions for performing any of the methods or steps provided herein.In some embodiments, a kit or composition can include a substrate withone or more capture probes comprising a spatial barcode and a capturedomain that captures a nucleic acid. In some instances, the methodsincludes means for sequencing the nucleic acid. In some instances, thekit includes reagents to detect and sequence the analyte. In someembodiments, the kit or composition further includes but is not limitedto one or more antibodies (and/or antigen-binding antibody fragments),labeled hybridization probes, primers, or any combination thereof forvisualizing one or more features of a tissue sample.

EXAMPLES Example 1: Efficient Analyte Capture from Slide-Mounted FreshFrozen Mouse Brain Sections onto Spatial Array Slides

Analyte capture onto spatially barcoded arrays and subsequent sequencingwas demonstrated under two-slide and control (not two-slide) conditions.For the two-slide condition, archived tissue-mounted on standard glassslides containing hematoxylin/eosin stained fresh frozen mouse brainsections were used. For the control condition, gene expression arrayslides with hematoxylin/eosin stained fresh frozen mouse brain sectionsmounted directly onto the array area were used. Under both conditions,tissue sections were subjected to a hematoxylin destaining step. Slidesprocessed according to the two-slide condition were briefly dried at 37°C., then mounted in an instrument along with a gene expression slide anda permeabilization buffer comprising sarkosyl and proteinase K. Uponinstrument closure (e.g., two-slides were positioned together in asandwich type of configuration), the tissue sections were permeabilizedfor 1 minute. For the tissue-mounted gene expression slides processedaccording to the control condition, sections were permeabilized for 5minutes using the same permeabilization buffer. For both conditions,following permeabilization, captured polyA-containing mRNA transcriptson the gene expression slides were reverse transcribed into cDNA,followed by standard sequencing library preparation and sequencing.

Results depicting median genes per spot and median UMI counts per spotare shown in FIG. 13 .

Visual heat map results showing Log 10 UMIs are shown in FIG. 14 .Spatial patterns of the Log 10 UMI counts were similar across thetwo-slide and control conditions.

Spatial clustering analysis (top row 1505) and analysis of hippocampaltranscript Hpca (bottom row 1510) are depicted in FIG. 15 . Spatialpatterns were comparable across the two-slide and control conditions.

Example 2: Method of Identifying Methylation Status of an Analyte

FIG. 10 is an exemplary workflow of the methods described herein foridentifying methylation status of a biological sample.

Specifically, a regular slide (e.g., a glass slide) is provided. Thebiological sample (e.g., a tissue) is placed on the slide, stained andimaged. The tissue is permeabilized, and a bisulfite reagent or adeaminating enzyme is applied to the tissue. During the deamination, thedouble-stranded DNA fragments become single-stranded due to loss ofparity when methylated cytosines are converted to uracils. After thebisulfite or deaminating reagent is washed away, one or more probestargeting regions of interest (such as CpG islands) on the analytes inthe tissue are hybridized to the analytes in the biological sample andare extended. The probes are designed to target (e.g., hybridize to)regions that do not have CG dinucleotides. They are also designed toinclude overhangs that specifically hybridize to the capture domain ofthe capture probes on a spatial array. After hybridization of the probesto the analyte, in this example one of the probes is extended using apolymerase, creating an oligonucleotide that is complementary to theregions between the hybridized probes. The probes are ligated, creatinga ligation product that includes a sequence complementary to thedeaminated analyte of interest. The ligation product (through one of theprobes) includes a sequence (e.g., an overhang sequence) complementaryto a capture probe on a spatial array.

As shown in the lower panel of FIG. 10 , a spatial array on a slidecomprising a plurality of capture probes with location UMIs is provided.The ligation product is transferred onto the spatial array slide using asuitable method described herein. Using the overhang of the ligationproduct, the ligation product hybridizes to the capture probes on thespatial array slide surface. The capture probes are extended using theligation product as a template. The ligation product is denatured, andusing a primer sequence from the capture probe, a plurality of nucleicacids are synthesized that include the sequence of the UMI and spatialbarcode, or a complement thereof, and the sequence of the region ofinterest on the analyte, or a complement thereof.

The plurality of nucleic acids is subsequently sequenced, and thesequence of all or a part of the UMI and spatial barcode, as well as thesequence of all or a part of the region of interest on the analyte isdetermined and analysis carried out to determine the % of methylatedfragments per target probe in a region of interest (HPCA genemethylation in this example).

Example 3: Method of Identifying Methylation Status in an Analyte UsingTagmented Samples

FIGS. 11A-11B is an exemplary workflow of the methods described herein.

As shown in FIG. 11A, the biological sample (e.g., a tissue section) isplaced on a regular slide without capture probes (the tissue isoptionally stained and imaged in this example). The tissue ispermeabilized, and the analytes (e.g., DNA) are tagmented by atransposase (Tn5) complexed with transposons that include methylatedadaptors, extended and ligated to create tagmented dsDNA. The tagmenteddouble-stranded DNA is deaminated using a bisulfite reagent or adeamination enzyme and denatured to form a single-stranded tagmentedDNA. The single-stranded tagmented DNA is then transferred to a spatialarray on a slide comprising a plurality of capture probes withlocational barcodes and UMIs. The transferred single-stranded tagmentedDNA is ligated to the capture probes on the slide using a splintoligonucleotide to bring the ends of the tagmented DNA in proximity tothe capture probe for ligation to occur. The captured DNA is thentreated with terminal transferase, dCTP and a uracil-friendly polymeraseto add a poly(C) tail to the captured DNA, which serves as ahybridization site for a poly(G) primer to form a plurality ofamplifiable nucleic acids.

As shown in FIG. 11B, these nucleic acids are prepared for sequencing,sequenced, and the sequence of all or a part of the UMI and spatialbarcode, as well as the sequence of all or a part of the region ofinterest on the analyte is determined. The data is analyzed and apercentage or degree of methylation of the DNA fragments is spatiallydetermined (HPCA gene methylation in this example).

Example 4: Method of Identifying Methylation Status of an Analyte

FIG. 16 is an exemplary workflow of the methods described herein.

Specifically, an array on a slide comprising a plurality of methylatedcapture probes (i.e., methylated cytosines) with location UMIs areprovided. Because the capture probes include methylated cytosines, thecapture probes are left intact in the presence of deaminating agents. Asshown in FIG. 16 , the sample (e.g., a tissue) is placed on a slide,stained and imaged. The tissue is permeabilized, and a bisulfite reagentor a deaminating enzyme is applied to the tissue. During thedeamination, the double-stranded DNA fragments become single-strandeddue to loss of parity when methylated cytosines are converted touracils. After the bisulfite or deaminating reagent is washed away, oneor more probes targeting regions of interest (such as CpG islands) onthe analytes in the tissue are hybridized to the analytes in thebiological sample and are extended. The probes are designed to target(e.g., hybridize to) regions that do not have CG dinucleotides. They arealso designed to include overhangs that specifically hybridize to thecapture domain of the capture probes on the slide surface. Afterhybridization of the probes to the analyte, in this example one of theprobes is extended using a polymerase, creating an oligonucleotide thatis complementary to the regions between the hybridized probes. Theprobes are ligated, creating a ligation product that includes a sequencecomplementary to the deaminated analyte of interest. The ligationproduct (through one of the probes) includes a sequence (e.g., anoverhang sequence) complementary to a capture probe on the array.

Using the overhang, the ligation product binds to the capture probes onthe slide surface. The capture probes are extended using the ligationproduct as a template. The ligation product is denatured, and using aprimer sequence from the capture probe, a plurality of nucleic acids aresynthesized that include the sequence of the UMI and spatial barcode, ora complement thereof, and the sequence of the region of interest on theanalyte, or a complement thereof.

These nucleic acids are subsequently sequenced, and the sequence of allor a part of the UMI and spatial barcode, as well as the sequence of allor a part of the region of interest on the analyte is determined andanalysis carried out to determine the % of methylated fragments pertarget probe in a region of interest (HPCA gene methylation in thisexample).

Example 5: Method of Identifying Methylation Status in an Analyte UsingTagmented Samples

FIG. 17 is an exemplary workflow of the methods described herein.

Specifically, an array on a slide comprising a plurality of methylatedcapture probes (i.e., methylated cytosines) with locational barcodes andUMIs are provided. Because the capture probes include methylatedcytosines, the capture probes are left intact in the presence ofdeaminating agents. As shown in FIG. 17 , the sample (e.g., a tissue) isplaced on a slide (optionally stained and imaged in this example). Thetissue is permeabilized, and the analytes (e.g., DNA) are tagmented by atransposase (Tn5 complexed with transposons that include methylatedadaptors), extended and ligated to create tagmented dsDNA. The tagmenteddouble-stranded DNA is denatured to form a single-stranded tagmentedDNA. The single-stranded tagmented DNA is ligated to the capture probeson the slide using a splint oligonucleotide to bring the ends of thetagmented DNA in proximity to the capture probe for ligation to occur.The ligation product is deaminated using a bisulfite reagent or adeamination enzyme. The captured DNA is then treated with terminaltransferase, dCTP and a uracil-friendly polymerase to add a poly(C) tailto the captured DNA, which serves as a hybridization site for a poly(G)primer to form a plurality of amplifiable nucleic acids. These nucleicacids are prepared for sequencing, sequenced, and the sequence of all ora part of the UMI and spatial barcode, as well as the sequence of all ora part of the region of interest on the analyte is determined. The datais then analyzed and a percentage or degree of methylation of the DNAfragments is determined (HPCA gene methylation in this example).

Example 6. Whole Genome Methylation Profiling Using Spatial GeneExpression Platform and in Droplets

FIGS. 18A-18B show an exemplary workflow of the tagmentation methodsdescribed herein.

Specifically, DNA methylation profiling is achieved on spatial geneexpression slides or droplets using transposase (e.g., Tn5, Tn7, Mu,Mariner, Sleeping Beauty, etc.) mediated tagmentation to add methylatedadapters or using an enzyme tethering approach for more targetedtagmentation, e.g. by fusing a transposase to an inactive restrictionenzyme like MspI (recognition site: CCGG, used in reduced representationbisulfite sequencing (RRBS) or to Dnmt1, the maintenancemethyltransferase).

As shown in FIG. 18A, analytes (e.g., DNA) in a biological sample arefragmented and tagged via tagmentation processes by, e.g., Tn5, MspI-Tn5fusion, or Dnmt1-Tn5 fusion. Methylated R2 handles are added to the 3′and/or 5′ ends of the DNA fragments.

The biological sample (e.g., tissue sample) is permeabilized and thetagmented DNA molecules bound to an array of capture probes on a geneexpression slide or a solid support in a droplet by ligating the handles(e.g., X2 or R2 handles) onto the DNA fragments and the capture probeson the slide surface or solid surface in the droplet, for example usinga splint oligonucleotide.

The ligated DNA fragments are extended using a dNTP mixed that includes5′ methyl CTP and the extended DNA fragments are denatured and removedfrom the slides or solid support in droplets using a denaturing reagent(e.g., KOH).

The denatured DNA fragments are treated with a deamination reagent(e.g., a bisulfite reagent). The handles and barcodes on the captureprobes are pre-methylated so they won't be affected by the deaminationprocess. For targeted capture, a probe panel is used to enrichdifferentially methylated regions (DMRs). Commercially available probepanels such as Agilent SureSelect XT Human Methyl-Seq panel (Agilent,Santa Clara, Calif.) can be used in the method described herein.

The deaminated DNA fragments are amplified using indexing PCR with asuitable polymerase such as Takara EpiTaq HS (Takara, Mountain View,Calif.). Optionally, the amplified DNA fragments are captured andmitochondrial DNA removed before sequencing.

Example 7: Method of Identifying Methylation Status in an Analyte

FIG. 19A shows an exemplary workflow used herein. In particular, a mousebrain tissue sample was sectioned and was placed on a microscopy slide.The tissue section 1901 was fixed, stained with H&E, and imaged. Afterdestaining with HCl, the sample was permeabilized for 15 minutes at roomtemperature with a protease in phosphate buffer saline (PBS). The tissuesection was treated with a solution comprising RNAse for 15 minutes atroom temperature in order to remove any RNA that could compete with DNAto hybridize to a capture probe on an array. The sample then was washingwith nuclease free water, and enzymatic deamination (New EnglandBiolabs® Inc.). Specifically, Tet methylcytosine dioxygenase 2 (TET2)was mixed in a solution to a 1× concentration, and the sample wasincubated for 1 hour at 37° C. The sample then was washed in 1×SSC.After, the sample was incubated in 20% formamide for 10 minutes at 85°C. The sample was treated with apolipoprotein B mRNA-editing enzyme,catalytic polypeptide (APOBEC) for 4 hours at 37° C. in order to convertdeaminating cytosine to uracil. After a wash step with 2×SSC, thenucleic acids in the sample were amplified 1903 using a DNA polymeraseand primers 1902 comprising a random hexamer (e.g., randomers) and apoly-thymine (n=30 thymine nucleotides) overhang. The amplified nucleicacids, shown in FIG. 19B and comprising a deaminated sequence, randomersequences, a poly-A tail, and a templated switching oligo (TSO), weredenatured with a solution of KOH for 5 minutes.

An array comprising captures probes having a capture domain with apoly-thymine sequence was sandwiched 1904 onto the sample. The tissuesection was permeabilized and the denatured nucleic acids were capturedon the array at 37° C. for 30 minutes. A DNA polymerase was used toextend 1905 the capture probe using the captured nucleic acid as atemplate. After, qPCR was performed; the resulting amplified productswere fragmented, indexed and sequenced 1906.

FIGS. 20 and 21 provide summaries of electropherograms for two samples.In FIG. 20 , the mouse brain tissue sample was deaminated after DNAamplification and 1× solid phase reversible immobilization (SPRI)cleanup, the elecropherogram demonstrating the range of amplificationproducts from the randomer based amplification as expected. FIG. 21shows an electropherogram after a 1:10 dilution of the final library,with additional SPRI clean up thereby focusing on a sequencing libraryof around 400 base pair fragments. The graphs in FIGS. 20 and 21 showthat sequences ranging from 400-700 base pairs were obtained viasequencing. Further, as shown in FIG. 22 , the sequencing data showed anincrease in adenine nucleotides and a decrease in cytosine and guanine.These data demonstrate a proof of concept that a biological sample canbe deaminated and nucleic acids from the biological sample can becaptured on a spatial array, sequenced and analyzed. Thus, using themethods here, one can spatially examine methylation patterns in abiological sample.

What is claimed is:
 1. A method for identifying a methylation status ofDNA in a biological sample, the method comprising: (a) contacting thebiological sample with a substrate; (b) providing one or moretransposomes to the biological sample, wherein the one or moretransposomes comprise one or more methylated adaptors, under conditionswherein the DNA is fragmented and one or more methylated adaptors fromthe one or more transposomes is inserted into the DNA, therebygenerating a plurality of tagmented DNA fragments, wherein a tagmentedDNA fragment of the plurality of tagmented DNA fragments comprises theone or more methylated adaptors; (c) ligating the tagmented DNA fragmentto a capture probe, thereby creating a ligation product; (d) deaminatingthe ligation product; and (e) determining (i) the sequence of a spatialbarcode of the capture probe or the complement thereof, and (ii) all orpart of the sequence of the ligation product, or a complement thereof,and using the determined sequences of (i) and (ii) to identify themethylation status of the DNA in the biological sample.
 2. The method ofclaim 1, wherein the substrate comprises an array comprising a pluralityof capture probes, wherein the capture probe is comprised in theplurality of capture probes, wherein the capture probe comprises thespatial barcode, and wherein if the capture probe comprises one or morecytosines, the one or more cytosines are methylated cytosines.
 3. Themethod of claim 1, further comprising, between steps (b) and (c):aligning the substrate with a second substrate comprising an array, suchthat at least a portion of the biological sample is aligned with atleast a portion of the array, wherein the array comprises a plurality ofcapture probes, wherein the capture probe is comprised in the pluralityof capture probes, wherein the capture probe comprises (i) the spatialbarcode and (ii) the capture domain; and when the biological sample isaligned with at least a portion of the array, releasing the tagmentedDNA fragment from the biological sample.
 4. The method of claim 3,wherein the substrate does not comprise an array comprising a pluralityof capture probes.
 5. The method of claim 3, wherein a 5′ end of thecapture probe is attached to the second substrate.
 6. The method ofclaim 1, wherein the ligating comprises enzymatic ligation or chemicalligation, and wherein the enzymatic ligation utilizes a ligase selectedfrom the group consisting of a PBCV-1 DNA ligase, a Chorella virus DNAligase, a single stranded DNA ligase, and a T4 DNA ligase.
 7. The methodof claim 1, wherein the DNA is genomic DNA.
 8. The method of claim 1,wherein the biological sample is a tissue section.
 9. The method ofclaim 1, wherein the biological sample is a fixed sample, a frozensample, a fresh sample, or a fresh frozen sample.
 10. The method ofclaim 1, wherein the biological sample is a cancer tissue sample. 11.The method of claim 1, wherein the deaminating comprises contacting thebiological sample with a composition comprising sodium bisulfite. 12.The method of claim 1, wherein the deaminating comprises treating thebiological sample with an enzyme.
 13. The method of claim 12, whereinthe enzyme is a cytidine deaminase or a demethylase.
 14. The method ofclaim 1, wherein the capture probe further comprises one or morefunctional domains, a unique molecular identifier, a cleavage domain, orcombinations thereof.
 15. The method of claim 1, further comprisingextending the capture probe using the ligation product to generate anextended ligation product.
 16. The method of claim 15, wherein theextending comprises incubating the ligation product with a terminaltransferase and a plurality of deoxycytidine triphosphate (dCTP)molecules.
 17. The method of claim 15, further comprising amplifying theextended ligation product.
 18. The method of claim 17, whereinamplifying the extended ligation product comprises using a primersequence that is complementary to a poly-cysteine sequence at the 3′ endof the extended ligation product.
 19. The method of claim 1, wherein thedetermining comprises sequencing (i) the spatial barcode, or acomplement thereof, and (ii) all or part of the ligation product, or acomplement thereof.
 20. The method of claim 1, wherein the one or moremethylated adaptors are inserted into the DNA using a transposasecomplexed with a transposon.
 21. The method of claim 20, wherein thetransposase is a Tn5 transposase or a functional derivative thereof, ora Tn7 transposase or a functional derivative thereof.
 22. The method ofclaim 1, wherein the ligating comprises use of a splint oligonucleotidecomprising (i) a sequence that hybridizes specifically to a portion ofthe capture probe; and (ii) a sequence that hybridizes specifically to aportion of the tagmented DNA fragment.
 23. The method of claim 22,wherein the splint oligonucleotide comprises a sequence that hybridizesspecifically to a portion of the one or more methylated adaptors. 24.The method of claim 1, wherein the one or more methylated adaptors is atleast about 10 nucleotides to about 50 nucleotides long.
 25. The methodof claim 1, wherein the one or more methylated adaptors is ligated to a5′ end of the fragmented DNA.
 26. The method of claim 1, wherein themethylated adaptor is ligated to a 3′ end of the fragmented DNA.
 27. Themethod of claim 1, further comprising imaging the biological sample. 28.The method of claim 27, wherein the imaging occurs prior to deaminatingthe biological sample.
 29. The method of claim 1, wherein the one ormore methylated adaptors comprises one or more methylated cytosines. 30.The method of claim 1, wherein identifying the methylation statusfurther comprises determining that about 1% to about 100% of cytosinesin the DNA comprises a methyl group.